Peptides

ABSTRACT

Isolated peptides that are fragments of protein products arising from frameshift mutations in genes associated with cancer are disclosed. The isolated peptides of the invention are capable of eliciting T cell immunity against cells harboring genes with such frameshift mutations. Cancer vaccines and therapeutically effective compositions containing the peptides of the invention are also described.

This application is a divisional application of application Ser. No.09/674,973, filed on Jun. 4, 2001, now U.S. Pat. No. 6,759,046 which wasfiled as PCT/NO92/00032 filed on May 3, 1999.

SUMMARY OF THE INVENTION

This invention relates to peptides which are fragments of proteinproducts arising from frameshift mutations in genes, which peptideselicit T cellular immunity, and to cancer vaccines and compositions foranticancer treatment comprising said peptides.

The invention further relates to a method for identifying such peptideswhich are fragments of protein products arising from frameshiftmutations in genes, which may elicit T cellular immunity which is usefulfor combating cancer associated with said mutated genes.

The invention also relates to DNA sequences encoding at least oneframeshift mutant peptide, and vectors comprising at least one insertionsite containing a DNA sequence encoding at least one frameshift mutantpeptide.

Further the invention relates to methods for the treatment orprophylaxis of cancers associated with frameshift mutations in genes byadministration of at least one frameshift mutant peptide or arecombinant virus vector comprising at least one insertion sitecontaining a DNA sequence encoding at least one frameshift mutantpeptide, or an isolated DNA sequence comprising a DNA sequence encodingat least one frameshift mutant peptide.

The present invention represents a further development of anticancertreatment or prophylaxis based on the use of peptides to generateactivation and strengthening of the anti cancer activity of the Tcellular arm of the body's own immune system.

TECHNICAL BACKGROUND

Tumour Antigens, Status:

T cell defined antigens have now been characterised in a broad spectrumof cancer types. These antigens can be divided into several main groups,depending on their expression. The two main groups are constituted bydevelopmental differentiation related antigens (tumour-testis antigens,oncofoetal antigens etc., such as MAGE antigens and CEA) and tissuespecific differentiation antigens (Tyrosinase, gp100 etc.). The groupcontaining the truly tumour specific antigens contains proteins that arealtered due to mutations in the genes encoding them. In the majority ofthese, the mutations are unique and have been detected in a single or ina small number of tumours. Several of these antigens seem to play a rolein oncogenesis.

Cancer Vaccines, Status:

The focus in cancer vaccine development has been on antigens expressedin a high degree within one form of cancer (such as melanoma) or in manykinds of cancers. One reason for this is the increased recruitment ofpatients into clinical protocols. The field is in rapid growth,illustrated by the accompanying table listing the cancer vaccineprotocols currently registered in the PDQ database of NCI.

Inheritable Cancer/Cancer Gene Testing:

Inherited forms of cancer occur at a certain frequency in thepopulation. For several of these cancer forms, the underlying geneticdefects have been mapped. This is also the case in Lynch syndromecancers which constitute an important group of inheritable cancer. Infamilies inflicted with this syndrome, family members inherit defectgenes encoding DNA Mismatch Repair (MMR) Enzymes. Carriers of such MMRdefects frequently develop colorectal cancer (HNPCC) and other forms ofcancer (list?). Mutations in MMR enzymes can be detected using genetesting in the same way as other cancer related genes can be detected.

Gene testing of risk groups in this case represents an ethical dilemma,since no acceptable forms for prophylactic treatment exist. At presentsurgery to remove the organ in danger to develop cancer has been theonly treatment option. In these patients, cancer vaccines will be a very(interesting) form of prophylaxis, provided efficient vaccines can bedeveloped.

The lack of efficient repair of mismatched DNA results in deletions andinsertions in one strand of DNA, and this happens preferentially instretches of DNA containing repeated units (repeat sequences). Untilnow, focus has been on repeat sequences in the form of non-codingmicrosattelite loci. Indeed microsattelite instability is the hallmarkof cancers resulting from MMR defects. We have taken another approach,and have concentrated on frameshift mutations occurring in DNA sequencescoding for proteins related to the oncogenic process. Such frameshiftmutations result in completely new amino acid sequences in theC-terminal part of the proteins, prematurely terminating where a novelstop codon appears. This results in two important consequences:

1) The truncated protein resulting from the frameshift is generallynonfunctional, in most cases resulting in “knocking out” of an importantcellular function. Aberrant proteins may also gain new functions such asthe capacity to aggragate and form plaques. In both cases the frameshiftresults in disease.

2) The short new C-terminal amino acid sequence resulting from the shiftin the reading frame (the “frameshift sequence”) is foreign to the body.It does not exist prior to the mutation, and it only exists in cellshaving the mutation, i.e. in tumour cells and their pre malignantprogenitors. Since they are completely novel and therefore foreign tothe immune system of the carrier, they may be recognised by T-cells inthe repertoire of the carrier. So far, nobody has focused on this aspectof frameshift mutations, and no reports exist on the characterisation offrameshift peptides from coding regions of proteins as tumour antigens.This concept is therefore novel and forms the basis for developingvaccines based on these sequences. It follows that such vaccines mayalso be used prophyllactively in persons who inherit defective enzymesbelonging to the MMR machinery. Such vaccines will therefore fill anempty space in the therapeutic armament against inherited forms ofcancer.

It has been shown that single amino acid substitutions in intracellular“self”-proteins may give rise to tumour rejection antigens, consistingof peptides differing in their amino acid sequence from the normalpeptide. The T cells which recognise these peptides in the context ofthe major histocompatibility (MHC) molecules on the surface of thetumour cells, are capable of killing the tumour cells and thus rejectingthe tumour from the host.

In contrast to antibodies produced by the B cells, which typicallyrecognise a free antigen in its native conformation and furtherpotentially recognise almost any site exposed on the antigen surface, Tcells recognise an antigen only if the antigen is bound and presented bya MHC molecule. Usually this binding will take place only afterappropriate antigen processing, which comprises a proteolyticfragmentation of the protein, so that the resulting peptide fragmentfits into the groove of the MHC molecule. Thereby T cells are enabled toalso recognise peptides derived from intracellular proteins. T cells canthus recognise aberrant peptides derived from anywhere in the tumourcell, in the context of MHC molecules on the surface of the tumour cell,and can subsequently be activated to eliminate the tumour cellharbouring the aberrant peptide.

M. Barinaga, Science, 257, 880–881, 1992 offers a short review of howMHC binds peptides. A more comprehensive explanation of the TechnicalBackground for this Invention may be found in D. Male et al, AdvancedImmunology, 1987, J.B.lippincott Company, Philadelphia. Both referencesare hereby included in their entirety.

The MHC molecules in humans are normally referred to as HLA (humanleukocyte antigen) molecules. They are encoded by the HLA region on thehuman chromosome No 6.

The HLA molecules appear as two distinct classes depending on whichregion of the chromosome they are encoded by and which T cellsubpopulations they interact with and thereby activate primarily. Theclass I molecules are encoded by the HLA A, B and C subloci and theyprimarily activate CD8+ cytotoxic T cells. The HLA class II moleculesare encoded by the DR, DP and DQ subloci and primarily activate CD4+ Tcells, both helper cells and cytotoxic cells.

Normally every individual has six HLA Class I molecules, usually twofrom each of the three groups A, B and C. Correspondingly, allindividuals have their own selection of HLA Class II molecules, againtwo from each of the three groups DP, DQ and DR. Each of the groups A,B, C and DP, DQ and DR are again divided into several subgroups. In somecases the number of different HLA Class I or II molecules is reduced dueto the overlap of two HLA subgroups.

All the gene products are highly polymorphic. Different individuals thusexpress distinct HLA molecules that differ from those of otherindividuals. This is the basis for the difficulties in finding HLAmatched organ donors in transplantations. The significance of thegenetic variation of the HLA molecules in immunobiology is reflected bytheir role as immune-response genes. Through their peptide bindingcapacity, the presence or absence of certain HLA molecules governs thecapacity of an individual to respond to peptide epitopes. As aconsequence, HLA molecules determine resistance or susceptibility todisease.

T cells may control the development and growth of cancer by a variety ofmechanisms. Cytotoxic T cells, both HLA class I restricted CD8+ and HLAClass II restricted CD4+, may directly kill tumour cells carrying theappropriate tumour antigens. CD4+ helper T cells are needed forcytotoxic CD8+ T cell responses as well as for antibody responses, andfor inducing macrophage and LAK cell killing.

A requirement for both HLA class I and II binding is that the peptidesmust contain a binding motif, which usually is different for differentHLA groups and subgroups. A binding motif is characterised by therequirement for amino acids of a certain type, for instance the onescarrying large and hydrophobic or positively charged side groups, indefinite positions of the peptide so that a narrow fit with the pocketsof the HLA binding groove is achieved. The result of this, takentogether with the peptide length restriction of 8–10 amino acids withinthe binding groove, is that it is quite unlikely that a peptide bindingto one type of HLA class I molecules will also bind to another type.Thus, for example, it may very well be that the peptide binding motiffor the HLA-A1 and HLA-A2 subgroups, which both belong to the class Igender, are as different as the motifs for the HLA-A1 and HLA-B1molecules.

For the same reasons it is not likely that exactly the same sequence ofamino acids will be located in the binding groove of the different classII molecules. In the case of HLA class II molecules the bindingsequences of peptides may be longer, and it has been found that theyusually contain from 10 to 16 amino acids, some of which, at one or bothterminals, are not a part of the binding motif for the HLA groove.

However, an overlap of the different peptide binding motifs of severalHLA class I and class II molecules may occur. Peptides that have anoverlap in the binding sequences for at least two different HLAmolecules are said to contain “nested T cell epitopes”. The variousepitopes contained in a “nested epitope peptide” may be formed byprocessing of the peptide by antigen presenting cells and thereafter bepresented to T cells bound to different HLA molecules. The individualvariety of HLA molecules in humans makes peptides containing nestedepitopes more useful as general vaccines than peptides that are onlycapable of binding to one type of HLA molecule.

Effective vaccination of an individual can only be achieved if at leastone type of HLA class I and/or II molecule in the patient can bind avaccine peptide either in it's full length or as processed and trimmedby the patient's own antigen presenting cells.

The usefulness of a peptide as a general vaccine for the majority of thepopulation increases with the number of different HLA molecules it canbind to, either in its full length or after processing by antigenpresenting cells.

In order to use peptides derived from a protein encoded by a mutatedgene as vaccines or anticancer agents to generate anti tumour CD4+and/or CD8+ T cells, it is necessary to investigate the mutant proteinin question and identify peptides that are capable, eventually afterprocessing to shorter peptides by the antigene presenting cells, tostimulate T cells.

PRIOR ART

In our International Application PCT/NO92/00032 (published asWO92/14756), the content of which is herein incorporated by reference,we described synthetic peptides and fragments of oncogene proteinproducts which have a point of mutation or translocations as compared totheir proto-oncogene or tumour suppressor gene protein. These peptidescorrespond to, completely cover or are fragments of the processedoncogene protein fragment or tumour suppressor gene fragment aspresented by cancer cells or other antigen presenting cells, and arepresented as a HLA-peptide complex by at least one allele in everyindividual. These peptides were also shown to induce specific T cellresponses to the actual oncogene protein fragment produced by the cellby processing and presented in the HLA molecule. In particular, wedescribed peptides derived from the P21 ras protein which had pointmutations at particular amino acid positions, namely position 12, 13 and61. These peptides have been shown to be effective in regulating thegrowth of cancer cells in vitro. Furthermore, the peptides were shown toelicit CD4+ T cell immunity against cancer cells harbouring the mutatedP21 ras oncogene protein through the administration of such peptides invaccination or cancer therapy schemes. Later we have shown that thesepeptides also elicit CD8+ T cell immunity against cancer cellsharbouring the mutated P21 ras onco gene protein through theadministration mentioned above.

However, the peptides described above will be useful only in certainnumber of cancers, namely those which involve oncogenes with pointmutations or translocation in a proto-oncogene or tumour suppressorgene. There is therefore a strong need for an anticancer treatment orvaccine which will be effective against a more general range of cancers.

In general, tumors are very heterogenous with respect to geneticalterations found in the tumour cells. This implies that both thepotential therapeutic effect and prophylactic strength of a cancervaccine will increase with the number of targets that the vaccine isable to elicit T cell immunity against. A multiple target vaccine willalso reduce the risk of new tumour formation by treatment escapevariants from the primary tumour.

DEFINITION OF PROBLEM SOLVED BY THE INVENTION

There is a continuing need for new anticancer agents based on antigenicpeptides giving rise to specific T cellular responses and toxicityagainst tumours and cancer cells carrying genes with mutations relatedto cancer. The present invention will contribute largely to supply newpeptides that can have a use in the combat and prevention of cancer asingredients in a multiple target anti-cancer vaccine.

Another problem solved by the present invention is that a protection ortreatment can be offered to the individuals belonging to family's orgroups with high risk for hereditary cancers. Hereditary cancers are inmany cases associated with genes susceptible to frameshift mutations asdescribed in this invention (i.e. mutations in mismatch repair genes).Today it is possible to diagnose risk of getting hereditary cancer butno pharmaceutical method for protection against the onset of the canceris available.

DEFINITION OF THE INVENTION

A main object of the invention is to obtain peptides corresponding topeptide fragments of mutant proteins produced by cancer cells which canbe used to stimulate T cells.

Another main object of the invention is to develop a cancer therapy forcancers based on the T cell immunity which may be induced in patients bystimulating their T cells either in vivo or in vitro with the peptidesaccording to the invention.

A third main object of the invention is to develop a vaccine to preventthe establishment of or to eradicate cancers based solely or partly onpeptides corresponding to peptides of the present invention which can beused to generate and activate T cells which produce cytotoxic T cellimmunity against cells harbouring the mutated genes.

A fourth main object of the invention is to design an anticancertreatment or prophylaxis specifically adapted to a human individual inneed of such treatment or prophylaxis, which comprises administering atleast one peptide according to this invention.

These and other objects of the invention are achieved by the attachedclaims.

Since frameshift mutations result in premature stop codons and thereforedeletion in large parts of the proteins, proteins with frameshiftmutations have generally not been considered to be immunogenetic andhave therefore not been considered as targets for immunotherapy. Thus ithas now surprisingly been found that a whole group of new peptidesresulting from frameshift mutations in tumour relevant genes are usefulfor eliciting T cell responses against cancer cells harbouring geneswith such frameshift mutations.

Genes containing a mono nucleoside base repeat sequence of at least fiveresidues, for example of eighth deoxyadenosine bases (AAAAAAA), or adi-nucleoside base repeat sequence of at least four di-nucleoside baseunits, for example of two deoxyadenosine-deoxycytosine units (ACAC), aresusceptible to frameshift mutations. The frameshift mutations occur,respectively, either by insertion of one or two of the mono-nucleosidebase residue or of one or two of the di-nucleoside base unit in therepeat sequence, or by deletion of one or two of the mono-nucleosidebase residue or of one or two of the di-nucleoside base unit from therepeat sequence. A gene with a frameshift mutation will from the pointof mutation code for a protein with a new and totally different aminoacid sequence as compared to the normal gene product. This mutantprotein with the new amino acid sequence at the carboxy end will bespecific for all cells carrying the modified gene.

In the remainder of this specification and claims the denominationframeshift mutant peptides will comprise such proteins and peptidefragments thereof.

It has now according to the present invention been found that such newprotein sequences arising from frameshift mutations in genes in cancercells give rise to tumour rejection antigens that are recognised by Tcells in the context of HLA molecules.

It has further according to the present invention been found a group ofpeptides corresponding to fragments of mutant proteins arising fromframeshift mutations in genes in cancer cells which can be used togenerate T cells. The said peptides can therefore also be used to rise aT cell activation against cancer cells harbouring a gene with aframeshift mutation as described above.

These peptides are at least 8 amino acids long and correspond, either intheir full length or after processing by antigen presenting cells, tothe mutant gene products or fragments thereof produced by cancer cellsin a human patient afflicted with cancer.

A peptide according to this invention is characterised in that it

-   -   a) is at least 8 amino acids long and is a fragment of a mutant        protein arising from a frameshift mutation in a gene of a cancer        cell; and    -   b) consists of at least one amino acid of the mutant part of a        protein sequence encoded by said gene; and    -   c) comprises 0–10 amino acids from the carboxyl terminus of the        normal part of the protein sequence preceding the amino terminus        of the mutant sequence and may further extend to the carboxyl        terminus of the mutant part of the protein as determined by a        new stop codon generated by the frameshift mutation in the gene;        and    -   d) induces, either in its full length or after processing by        antigen presenting cell, T cell responses.

The peptides of this invention contain preferably 8–25, 9–20, 9–16, 8–12or 20–25 amino acids. They may for instance contain 9, 12, 13, 16 or 21amino acids.

It is most preferred that the peptides of the present invention are atleast 9 amino acids long, for instance 9–18 amino acids long, but due tothe processing possibility of the antigen presenting cells also longerpeptides are very suitable for the present invention. Thus the wholemutant amino acid sequence may be used as a frameshift mutant peptideaccording to the present invention, if it comprises 8 amino acids ormore.

The invention further relates to a method for vaccination of a persondisposed for cancer, associated with a frameshift mutation in a gene,consisting of administering at least one peptide of the invention one ormore times in an amount sufficient for induction of T-cell immunity tothe mutant proteins encoded by the frameshift mutated gene.

The invention also relates to a method for treatment of a patientafflicted with cancer associated with frameshift mutation in genes,consisting of administering at least one peptide of the invention one ormore times in an amount sufficient for induction of T-cell immunity tomutant proteins arising from frameshift mutations in the genes of cancercells.

Furthermore, it has according to the present invention been found amethod for identifying new peptides which correspond to fragments ofproteins arising from frameshift mutations in genes. This method ischaracterised by the following steps:

-   -   1) identifying a gene in a cancer cell susceptible to frameshift        mutation by having a mono nucleoside base repeat sequence of at        least five residues, or a di-nucleoside base repeat sequence of        at least four di-nucleoside base units; and    -   2) removing, respectively, one nucleoside base residue or one        di-nucleoside base unit from the repeat sequence and identifying        the amino acid sequence of the protein encoded by the altered        gene sequence as far as to include a new stop codon; and/or    -   3) removing, respectively, two nucleoside base residues or two        di-nucleoside base units from the repeat sequence and        identifying the amino acid sequence of the protein encoded by        the altered gene sequence as far as to include a new stop codon;        and/or    -   4) inserting, respectively, one nucleoside base residue or one        di-nucleoside base unit in the repeat sequence and identifying        the amino acid sequence of the protein encoded by the altered        gene sequence as far as to include a new stop codon; and/or    -   5) inserting, respectively, two nucleoside base residues or two        di-nucleoside base units in the repeat sequence and identifying        the amino acid sequence of the protein encoded by the altered        gene sequence as far as to include a new stop codon.

In order to determine whether the peptides thus identified are useablein the compositions and methods according to the present invention forthe treatment or prophylaxis of cancer, the following further stepshould be performed:

-   -   6) determining whether the new peptide, either in their full        length or as shorter fragments of the peptides, are able to        stimulate T cells.

Optionally a further step may be added as follows:

-   -   7) determining peptides containing nested epitopes for different        major HLA class I and/or HLA class II molecules.

DETAILED DESCRIPTION OF THE INVENTION

In the present description and claims, the amino acids are representedby their one letter abbreviation as known in the art.

The peptides of the present invention shall be explicitly exemplifiedthrough two different embodiments, wherein cancer develops based onframeshift mutations in specific genes, namely the BAX gene and TGFβRIIgene:

I) BAX Gene

It has been established that the BAX gene is involved in regulation ofsurvival or death of cells by promoting apoptosis. The human BAX genecontains a repeat sequence of eight deoxyguanosine bases (G8) in thethird exon, spanning codons 38 to 41 (ATG GGG GGG GAG).

Frameshift mutations in this G8 repeat have been observed, both as G7(ATG GGG GGG AGG) and G9 (ATG GGG GGG GGA) repeats, both in colon cancercells and prostate cancer cells. The occurency is more than 50% of theexamined cases (Rampino, N. et al., “Somatic frameshift mutations in theBAX gene in colon cancers of the microsatellite mutator phenotype.”,Science (Washington D.C.), 275: 967–969, 1997). The modified BAX geneproducts are unable to promote apoptosis and thus makes further tumourprogress possible. Furthermore the modified gene products are only foundin cancer cells and are therefore targets for specific immunotherapy.

According to the present invention, peptides corresponding to thetransformed BAX protein products arising from frameshift mutations inthe BAX gene can be used as anticancer therapeutical agents or vaccineswith the function to trigger the cellular arm of the immune system(T-cells) against cancer cells in patients afflicted with cancersassociated with a modified BAX gene.

Frameshift mutations in the BAX gene result in mutant peptide sequenceswith the first amino acid of the altered sequence in position 41 ascompared to the normal BAX protein (Table 1, seq.id. no. 1 to 4).

TABLE 1 amino acid pos 41 51 61 71 normal bax peptide; EAPELALDPVPQDASTKKLS ECLKRIGDEL DS . . . (bax − 1G); RHPSWPWTRC LRMRPPRS seq. id.no. 1 (bax + 2G); GRHPSWPWTR CLRMRPPRS seq. id. no. 4 (bax − 2G);GTRAGPGPGA SGCVHQEAER VSQAHRGRTG Q seq. id. no. 2 (bax + 1G); GGTRAGPGPGASGCVHQEAE RVSQAHRGRT GQ seq. id. no. 3

Table 2 shows one group of peptides according to the present invention:

TABLE 2 IQDRAGRMGGRHPSWPWTRCLRMRPPRS: seq. id. no. 5IQDRAGRMGGGRHPSWPWT: seq. id. no. 6IQDRAGRMGGGGTRAGPGPGASGCVHQEAERVSQAHRGRTGQ: seq. id. no. 7IQDRAGRMGGGTRAGPGPG: seq. id. no. 8

The peptides listed in Table 3 were used for in vitro generation of Tcells that recognise mutant BAX peptides.

TABLE 3 RHPSWPWTRCLRMRPPRS: seq id no 1 IQDRAGRMGGRHPSWPWTRCLR: seq idno 9 IQDRAGRMGGGRHPSWPWT: seq id no 6 ASGCVHQEAERVSQAHRGRTGQ: seq id no10 GGTRAGPGPGASGCVHQEAERV: seq id no 11 IQDRAGRMGGGGTRAGPGPGAS: seq idno 12 IQDRAGRMGGGTRAGPGPG: seq id no 8

The most preferred peptides according to this embodiment of the presentinvention are listed in Table 4:

TABLE 4 RHPSWPWTRCLRMRPPRS: seq id no 1 GTRAGPGPGASGCVHQEAERVSQAHRGRTGQ:seq id no 2 GGTRAGPGPGASGCVHQEAERVSQAHRGRTGQ: seq id no 3GRHPSWPWTRCLRMRPPRS: seq id no 4 IQDRAGRMGGRHPSWPWTRCLRMRPPRS: seq. id.no. 5 IQDRAGRMGGGRHPSWPWT: seq. id. no. 6IQDRAGRMGGGGTRAGPGPGASGCVHQEAERVSQAHRGRTGQ: seq. id. no. 7IQDRAGRMGGGTRAGPGPG: seq id no 8 IQDRAGRMGGRHPSWPWTRCLR: seq id no 9ASGCVHQEAERVSQAHRGRTGQ: seq id no 10 GGTRAGPGPGASGCVHQEAERV: seq id no11 IQDRAGRMGGGGTRAGPGPGAS: seq id no 122) TGFβRII

It has been established that the TGFβRII gene is involved in regulationof cell growth. TGFβRII is a receptor for TGFβ which down regulates cellgrowth. The human gene coding for TGFβRII contains a repeat sequence often deoxyadenosine bases (A10) from base no. 709 to base no. 718 (GAAAAA AAA AAG CCT). In colon cancers and pancreatic cancers frameshiftmutations in this A10 repeat have been observed, both as A9 (GAA AAA AAAAGC CT) and A11 (GAA AAA AAA AAA GCC) repeats, in approximately 80% ofthe examined cases (Yamamoto, H., “Somatic frameshift mutations in DNAmismatch repair and proapoptosis genes in hereditary nonpolyposiscolorectal cancer.”, Cancer Research 58, 997–1003, Mar. 1, 1998). Themodified TGFβRII gene products are unable to bind TGFβ and the signalfor down regulation of cell growth is eliminated and thus makes furthertumour progress possible. Furthermore the modified gene products areonly found in cancer cells and are therefore targets for immunotherapy.

Consequently peptides corresponding to the transformed. TGFβRII proteinproducts arising from frameshift mutations in the TGFβRII gene can beused as anticancer therapeutical agents or vaccines with the function totrigger the cellular arm of the immune system (T-cells) against cancercells in patients afflicted with cancers associated with a modifiedTGFβRII gene.

Frameshift mutations in the TGFβRII gene result in mutant peptidesequences with the first amino acid of the altered sequence in eitherPosition 133 (one and two base deletions) or 134 (one and two baseinsertions) as compared to the normal TGFβRII protein (Table 5,seq.id.nos. 13 and 21).

TABLE 5 amino acid pos. 133 normal TGFβRII; K PGETFFMCSC SSDECNDNIIFSEEYNTSNP DLLL (−1A); S LVRLSSCVPV ALMSAMTTSS SQKNITPAIL TCC seq id no13 (+2A); SLVRLSSCVP VALMSAMTTS SSQKNITPAI LTCC seq id no 13 TGFbRII +1A); AW TGFbRII − 2A); A W

Table 6 shows one groups of peptides of this invention:

TABLE 6 SPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC: seq id no 14PKCIMKEKKKSLVRLSSCV: seq id no 15 SPKCIMKEKKAW: seq id no 19PKCIMKEKKKAW: seq id no 20

Table 7 presents peptides that were used for in vitro generation of Tcells that recognise mutant TGFβRII peptides.

TABLE 7 PKCIMKEKKKSLVRLSSCV: seq id no 15 ALMSAMTTSSSQKNITPAILTCC: seqid no 16 SLVRLSSCVPVALMSAMTTSSSQ: seq id no 17 SPKCIMKEKKSLVRLSSCVPVA:seq id no 18 SPKCIMKEKKAW: seq id no 19 PKCIMKEKKKAW: seq id no 20AMTTSSSQKNITPAILTCC: seq id no 21 SLVRLSSCV: seq id no 428

The most preferred peptides of this embodiment of the present inventionare:

TABLE 8 SLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC: seq id no 13SPKCIMKEKKSLVRLSSCVPVALMSAMTTSSSQKNITPAILTCC: seq id no 14PKCIMKEKKKSLVRLSSCV: seq id no 15 ALMSAMTTSSSQKNITPAILTCC: seq id no 16SLVRLSSCVPVALMSAMTTSSSQ: seq id no 17 SPKCIMKEKKSLVRLSSCVPVA: seq id no18 SPKCIMKEKKAW: seq id no 19 PKCIMKEKKKAW: seq id no 20AMTTSSSQKNITPAILTCC: seq id no 21 SLVRLSSCV: seq id no 428

Other peptides of the invention can be fragments of the peptides listedin the Tables 1–8 above. Such fragments are most preferred from 9–16amino acids long and include at least one amino acid from the mutantpart of the protein.

As used in this description and claims the term fragment is intended tospecify a shorter part of a longer peptide or of a protein.

Other cancer associated genes containing repeat sequences of anucleoside base and which therefore are susceptible to frameshiftmutations and consequently are potential candidates for peptidesaccording to the present invention (seq id nos according to table 9 aregiven in parentheses in each case) are the following:

-   Human TGF-β-2 (hTGFβ2) gene (seq id nos 22–29)-   Deleted in colorectal cancer (DCC) gene (seq.id.nos. 30–34)-   Human breast and ovarian cancer susceptibility (BRCA1) gene    (seq.id.nos. 378–387)-   Human breast cancer susceptibility (BRCA2) gene (seq.id.nos. 35–94)-   Human protein tyrosine phosphatase (hPTP) gene (seq.id.nos. 95–102)-   Human DNA topoisomerase II (top2) gene (seq.id.nos. 103–108)-   Human kinase (TTK) gene (seq.id.nos. 109–120)-   Human transcriptional repressor (CTCF) gene (seq.id.nos. 121–127)-   Human FADD-homologous ICE/CED-3-like protease gene (seq.id.nos.    128–133)-   Human putative mismatch repair/binding protein (hMSH3) gene    (seq.id.nos. 134–147)-   Human retinoblastoma binding protein 1 isoform I (hRBP1) gene    (seq.id.nos. 148–156)-   Human FMR1 (hFMR1) gene (seq.id.nos. 157–161)-   Human TINUR gene (seq.id.nos. 162–169) b-raf oncogene (seq.id.nos.    170–175)-   Human neurofibromin (NF1) gene (seq.id.nos. 176–181)-   Human germline n-myc gene (seq.id.nos. 182–188)-   Human n-myc gene (seq.id.nos. 189–194)-   Human ras inhibitor gene (seq.id.nos. 195–199)-   Human hMSH6 gene (seq.id.nos. 200–203 and 293–297)-   Human nasopharynx carcinoma EBV BNLF-1 gene (seq.id.nos. 204–210)-   Human cell cycle regulatory protein (E1A-binding protein) p300 gene    (seq.id.nos. 211–218)-   Human B-cell lymphoma 3-encoded protein (bcl-3) gene (seq.id.nos.    219–226)-   Human transforming growth factor-beta induced gene product (BIGH3)    (seq.id.nos. 227–232)-   Human transcription factor ETV1 gene. (seq.id.nos. 233–239)-   Human insulin-like growth factor binding protein (IGFBP4) gene    (seq.id.nos. 240–246)-   Human MUC1 gene (seq.id.nos. 247–266)-   Human protein-tyrosine kinase (JAK1) gene (seq.id.nos. 267–271)-   Human protein-tyrosine kinase (JAK3) gene (seq.id.nos. 272–279)-   Human Flt4 gene (for transmembrane tyrosinase kinase) (seq.id.nos.    280–284)-   Human p53 associated gene (seq.id.nos. 285–292)-   Human can (hCAN) gene (seq.id.nos. 298–300)-   Human DBL (hDBL) proto-oncogene/Human MCF2PO (hMCF2PO) gene    (seq.id.nos. 301–306)-   Human dek (hDEK) gene (seq.id.nos. 307–309)-   Human retinoblastoma related protein (p107) gene (seq.id.nos.    310–313)-   Human G protein-coupled receptor (hGPR1) gene (seq.id.nos. 314–319)-   Human putative RNA binding protein (hRBP56) gene (seq.id.nos.    320–325)-   Human transcription factor (hITF-2) gene (seq.id.nos. 326–327)-   Human malignant melanoma metastasis-supressor (hKiSS-1) gene    (seq.id.nos. 328–334)-   Human telomerase-associated protein TP-1 (hTP-1) gene (seq.id.nos.    335–348)-   Human FDF-5 (hFDF-5) gene (seq.id.nos. 349–356)-   Human metastasis-assosiated mta1 (hMTA1) gene (seq.id.nos. 357–362)-   Human transcription factor TFIIB 90 kDa subunit (hTFIIB90) gene (seq    id nos 363–369)-   Human tumour suppressor (hLUCA-1) gene (seq id nos 370–377)-   Human Wilm's tumour (WIT-1) associated protein (seq id nos 388–393)-   Human cysteine protease (ICErel-III) gene (seq id nos 394–398 and    459)-   Human Fas ligand (FasL) gene (seq id nos 399–403)-   Human BRCA1-associated RING domain protein (BARD1) gene (seq id nos    404–417)-   Human mcf.2 (hMCF.2) gene (seq id nos 418–422)-   Human Fas antigen (fas) gene (seq id nos 423–427)-   Human DPC4 gene (seq id nos 429–437).

The mutant peptides that are the results of frameshift mutation in thesegenes, in accordance with the present invention, are listed in table 9.

TABLE 9 TVGRPHISC; seq id no 22 KTVGRPHISC; seq id no 23KQWEDPTSPANVIALLQT; seq id no 24 QWEDPTSPANVIALLQT; seq id no 25QKTIKSTRKKTVGRPHISC; seq id no 26 QKTIKSTRKKKTVGRPHISC; seq id no 27QKTIKSTRKKKQWEDPTSPANVIALLQT; seq id no 28 QKTIKSTRKKQWEDPTSPANVIALLQT;seq id no 29 AADLQQQFVHFLDCWDVSSIPFTLHLPQAQDITT; seq id no 30 GKDAKEKSS;seq id no 31 GKDAKEKKSS; seq id no 32GKDAKEKKAADLQQQFVHFLDCWDVSSIPFTLHLPQA seq id no 33 QDITT;GKDAKEKAADLQQQFVHFLDCWDVSSIPFTLHLPQAQ seq id no 34 DITT;FSMKQTLMNVKNLKTK; seq id no 35 KFSMKQTLMNVKNLKTK; seq id no 36VRTSKTRKKFSMKQTLMNVKNLKTK; seq id no 37 VRTSKTRKKKFSMKQTLMNVKNLKTK; seqid no 38 VRTSKTRKKNFP; seq id no 39 VRTSKTRKNFP; seq id no 40IKKKLLQFQK; seq id no 41 KIKKKLLQFQK; seq id no 42 KSRRNYFNFKNNCQSRL;seq id no 43 SRRNYFNFKNNCQSRL; seq id no 44 TNLRVIQKIKKKLLQFQK; seq idno 45 TNLRVIQKKIKKKLLQFQK; seq id no 46 TNLRVIQKKSRRNYFNFKNNCQSRL; seqid no 47 TNLRVIQKSRRNYFNFKNNCQSRL; seq id no 48 KIMIT; seq id no 49NIDKIPEKIMIT; seq id no 50 NIDKIPEKKIMIT; seq id no 51 IINAN; seq id no52 KIINAN; seq id no 53 NDKTVSEKIINAN; seq id no 54 NDKTVSEKKIINAN; seqid no 55 NGLEKEYLMVNQKE; seq id no 56 SQTSLLEAKNGLEKEYLMVNQKE; seq id no57 SQTSLLEAKKNGLEKEYLMVNQKE; seq id no 58 SQTSLLEAKKMA; seq id no 59SQTSLLEAKMA; seq id no 60 TLVFPK; seq id no 61 KTLVFPK; seq id no 62LKNVEDQKTLVFPK; seq id no 63 LKNVEDQKKTLVFPK; seq id no 64 LKNVEDQKKH;seq id no 65 LKNVEDQKH; seq id no 66 KKIQLY; seq id no 67 KKKIQLY; seqid no 68 RKRFSYTEYLASIIRFIFSVNRRKEIQNLSSCNFKI; seq id no 69LRIVSYSKKKKIQLY; seq id no 70 LRIVSYSKKKKKIQLY; seq id no 71LRIVSYSKKRKRFSYTEYLASIIRFIFSVNRRKEIQ- seq id no 72 -NLSSCNFKI;LRIVSYSKRKRFSYTEYLASIIRFIFSVNRRKEIQN- seq id no 73 -LSSCNFKI;QDLPLSSICQTIVTIYWQ; seq id no 74 KQDLPLSSICQTIVTIYWQ; seq id no 75NRTCPFRLFVRRMLQFTGNKVLDRP; seq id no 76 GFVVSVVKKQDLPLSSICQTIVTIYWQ; seqid no 77 GFVVSVVKKKQDLPLSSICQTIVTIYWQ; seq id no 78GFVVSVVKKNRTCPFRLFVRRMLQFTGNKVLDRP; seq id no 79GFVVSVVKNRTCPFRLFVRRMLQFTGNKVLDRP; seq id no 80 YRKTKNQN; seq id no 81KYRKTKNQN; seq id no 82 NTERPKIRTN; seq id no 83 DETFYKGKKYRKTKNQN; seqid no 84 DETFYKGKKKYRKTKNQN; seq id no 85 DETFYKGKKNTERPKIRTN; seq id no86 DETFYKGKNTERPKIRTN; seq id no 87 LSINNYRFQMKFYFRFTSHGSPFTSANF; seq idno 88 KLSINNYRFQMKFYFRFTSHGSPFTSANF; seq id no 89 NSVSTTTGFR; seq id no90 NIQLAATKKLSINNYRFQMKFYFRFTSHGSPFTSAN seq id no 91 F;NIQLAATKKKLSINNYRFQMKFYFRFTSHGSPFTSA seq id no 92 NF;NIQLAATKKNSVSTTTGFR; seq id no 93 NIQLAATKNSVSTTTGFR; seq id no 94MEHVAPGRMSASPQSPTQ; seq id no 95 KMEHVAPGRMSASPQSPTQ; seq id no 96KWSTWLQAECQHLHSPQRSDKPQQAGLDQQHHCFAL- seq id no 97-DSSPGPRPVFLQLLGLMGQGRHD; WSTWLQAECQHLHSPQRSDKPQQAGLDQQHHCFALD- seq idno 98 -SSPGPRPVFLQLLGLMGQGRHD; TFSVWAEKMEHVAPGRMSASPQSPTQ; seq id no 99TFSVWAEKKMEHVAPGRMSASPQSPTQ; seq id no 100TFSVWAEKKWSTWLQAECQHLHSPQRSDKPQQAGLD- seq id no 101-QQHHCFALDSSPGPRPVFLQLLGLMGQGRHD; TFSVWAEKWSTWLQAECQHLHSPQRSDKPQQAGLDQ-seq id no 102 -QHHCFALDSSPGPRPVFLQLLGLMGQGRHD; HKWLKFCLLRLVKESFHE; seqid no 103 KHKWLKFCLLRLVKESFHE; seq id no 104KGGKAKGKKHKWLKFCLLRLVKESFHE; seq id no 105 KGGKAKGKKKHKWLKFCLLRLVKESFHE;seq id no 106 KGGKAKGKKNTNG; seq id no 107 KGGKAKGKNTNG; seq id no 108VNNFFKKL; seq id no 109 KVNNFFKKL; seq id no 110 LSQGNVKKVNNFFKKL; seqid no 111 LSQGNVKKKVNNFFKKL; seq id no 112GEKNDLQLFVMSDRRYKIYWTVILLNPCGNLHLKTTS seq id no 113 L;KGEKNDLQLFVMSDRRYKIYWTVILLNPCGNLHLKTT seq id no 114 SL; KGKKMICSYS; seqid no 115 GKKMICSYS; seq id no 116 SSKTFEKKGEKNDLQLFVMSDRRYKIYWTVILLNPC-seq id no 117 -GNLHLKTTSL; SSKTFEKKKGEKNDLQLFVMSDRRYKIYWTVILLNP- seq idno 118 -CGNLHLKTTSL; SSKTFEKKKGKKMICSYS; seq id no 119SSKTFEKKGKKMICSYS; seq id no 120 QRKPKRANCVIQRRAKM; seq id no 121KQRKPKRANCVIQRRAKM; seq id no 122 NKENQKEQTALLYRGGQRCRCVCLRF; seq id no123 NKENQKEQTALLYRGGQRCRCVCLRF; seq id no 123PDYQPPAKKQRKPKRANCVIQRRAKM; seq id no 124 PDYQPPAKKKQRKPKRANCVIQRRAKM;seq id no 125 PDYQPPAKKNKENQKEQTALLYRGGQRCRCVCLRF; seq id no 126PDYQPPAKNKENQKEQTALLYRGGQRCRCVCLRF; seq id no 127 NLSSLLI; seq id no 128TCLPF; seq id no 129 QPTFTLRKNLSSLLI; seq id no 130 QPTFTLRKKNLSSLLI;seq id no 131 QPTFTLRKKTCLPF; seq id no 132 QPTFTLRKTCLPF; seq id no 133RATFLLSLWECSLPQARLCLIVSRTGLLVQS; seq id no 134 GQHFYWHCGSAACHRRGCV; seqid no 135 KENVRDKKRATFLLSLWECSLPQARLCLIVSRTGLLV seq id no 136 QS;KENVRDKKKRATFLLSLWECSLPQARLCLIVSRTGLL seq id no 137 VQS;KENVRDKKKGQHFYWHCGSAACHRRGCV; seq id no 138 KENVRDKKGQHFYWHCGSAACHRRGCV;seq id no 139 ITHTRWGITTWDSWSVRMKANWIQAQQNKSLILSPSF seq id no 140 TK;KITHTRWGITTWDSWSVRMKANWIQAQQNKSLILSPS seq id no 141 FTK;KLLTPGGELPHGILGQ; seq id no 142 LLTPGGELPHGILGQ; seq id no 143PPVCELEKITHTRWGITTWDSWSVRMKANWIQAQQN- seq id no 144 -KSLILSPSFTK;PPVCELEKKITHTRWGITTWDSWSVRMKANWIQAQQ- seq id no 145 -NKSLILSPSFTK;PPVCELEKKLLTPGGELPHGILGQ; seq id no 146 PPVCELEKLLTPGGELPHGILGQ; seq idno 147 SLKDELEKMKI; seq id no 148 SLKDELEKKMKI; seq id no 149LGQSSPEKKNKN; seq id no 150 LGQSSPEKNKN; seq id no 151RLRRINGRGSQIRSRNAFNRSEE; seq id no 152 EPKVKEEKKT; seq id no 153EPKVKEEKKKT; seq id no 154 EPKVKEEKKRLRRINGRGSQIRSRNAFNRSEE; seq id no155 EPKVKEEKRLRRINGRGSQIRSRNAFNRSEE; seq id no 156 TFRYKGKQHPFFST; seqid no 157 GPNAPEEKNH; seq id no 158 GPNAPEEKKNH; seq id no 159GPNAPEEKKTFRYKGKQHPFFST; seq id no 160 GPNAPEEKTFRYKGKQHPFFST; seq id no161 MQNTCV; seq id no 162 KMQNTCV; seq id no 163 KCKIRVFSK; seq id no164 CKIRVFSK; seq id no 165 FFKRTVQKMQNTCV; seq id no 166FFKRTVQKKMQNTCV; seq id no 167 FFKRTVQKKCKIRVFSK; seq id no 168FFKRTVQKCKIRVFSK; seq id no 169 LPHYLAH; seq id no 170 CLITWLTN; seq idno 171 GSTTGLSATPLPHYLAH; seq id no 172 GSTTGLSATPPLPHYLAH; seq id no173 GSTTGLSATPPCLITWLTN; seq id no 174 GSTTGLSATPCLITWLTN; seq id no 175RFADKPRPN; seq id no 176 DLPTSPDQTRSGPVHVSVEP; seq id no 177DSAAGCSGTPRFADKPRPN; seq id no 178 DSAAGCSGTPPRFADKPRPN; seq id no 179DSAAGCSGTPPDLPTSPDQTRSGPVHVSVEP; seq id no 180DSAAGCSGTPDLPTSPDQTRSGPVHVSVEP; seq id no 181AHPETPAQNRLRIPCSRREVRSRACKPPGAQGSDER- seq id no 182 -RGKASPGRDCDVRTGRP;PAHPETPAQNRLRIPCSRREVRSRACKPPGAQGSDE- seq id no 183 -RRGKASPGRDCDVRTGRP;RPTRRHPRRIASGSPAVGGR; seq id no 184VAIRGHPRPPAHPETPAQNRLRIPCSRREVRSRACK- seq id no 185-PPGAQGSDERRGKASPGRDCDVRTGRP; VAIRGHPRPPPAHPETPAQNRLRIPCSRREVRSRAC- seqid no 186 -KPPGAQGSDERRGKASPGRDCDVRTGRP; VAIRGHPRPPRPTRRHPRRIASGSPAVGGR;seq id no 187 VAIRGHPRPRPTRRHPRRIASGSPAVGGR; seq id no 188RGRTSGRSLSCCRRPRCRPAVASRSTAPSPRAGSR- seq id no 189-RCCLRTSCGAARPRRTRSACGDWVASPPTRSS- -SRTACGAASPPARSWSAP; GGGHLEEV; seq idno 190 YFGGPDSTPRGRTSGRSLSCCRRPRCRPAVASR- seq id no 191-STAPSPRAGSRRCCLRTSCGAARPRRTRSACGD- -WVASPPTRSSSRTACGAASPPARSWSAP;YFGGPDSTPPRGRTSGRSLSCCRRPRCRPAVASR- seq id no 192-STAPSPRAGSRRCCLRTSCGAARPRRTRSACGDW- -VASPPTRSSSRTACGAASPPARSWSAP;YFGGPDSTPPGGGHLEEV; seq id no 193 YFGGPDSTPGGGHLEEV; seq id no 194HRVADP; seq id no 195 LSQSSELDPPSSR; seq id no 196 LSQSSELDPPPSSR; seqid no 197 LSQSSELDPPHRVADP; seq id no 198 LSQSSELDPHRVADP; seq id no 199VILLPEDTPPS; seq id no 200 VILLPEDTPPPS; seq id no 201 VILLPEDTPPLLRA;seq id no 202 VILLPELDPLLRA; seq id no 203 PSPLP; seq id no 204PLLFHRPCSPSPALGATVLAVYRYE; seq id no 205 LLFHRPCSPSPALGATVLAVYRYE; seqid no 206 APRPPLGPPSPLP; seq id no 207 APRPPLGPPPSPLP; seq id no 208APRPPLGPPPLLFHRPCSPSPALGATVLAVYRYE; seq id no 209APRPPLGPPLLFHRPCSPSPALGATVLAVYRYE; seq id no 210TQVLPQGCSLSLLHTTFPHRQVPHILDW; seq id no 211PTQVLIPQGCSLSLLHTTFPHRQVPHILDW; seq id no 212PLQSFPKDAASAFSTPRFPTDKFPTSWTGSCPGQPH- seq id no 213 -GTRAFCQPGPEFNAFSAC;LQSFPKDAASAFSTPRFPTDKFPTSWTGSCPGQPHG- seq id no 214 -TRAFCQPGPEFNAFSAC;PSPRPQSQPPTQVLPQGCSLSLLHTTFPHRQVPHILD seq id no 215 W;PSPRPQSQPPPTQVLPQGCSLSLLHTTFPHRQVPHIL seq id no 216 DW;PSPRPQSQPPPLQSFPKDAASAFSTPRFPTDKFPTS- seq id no 217-WTGSCPGQPHGTRAFCQPGPEFNAFSAC; PSPRPQSQPPLQSFPKDAASAFSTPRFPTDKFPTS- seqid no 218 -WTGSCPGQPHGTRAFCQPGPEFNAFSAC; TAWPGRRRFTTPEPYCLCTPLGPWAPRFLW;seq id no 219 PTAWPGRRRFTTPEPYCLCTPLGPWAPRFLW; seq id no 220PRPGPAGGALLPRSLTAFVPHSGHGLPVSSGEPAYT- seq id no 221 -PIPHDVPHGTPPFC;RPGPAGGALLPRSLTAFVPHSGHGLPVSSGEPAYTP- seq id no 222 -IPHDVPHGTPPFC;DLPAVPGPPTAWPGRRRFTTPEPYCLCTPLGPWAPRF seq id no 223 LW;DLPAVPGPPPTAWPGRRRFTTPEPYCLCTPLGPWAPR seq id no 224 FLW;DLPAVPGPPPRPGPAGGALLPRSLTAFVPHSGHGLP- seq id no 225-VSSGEPAYTPIPHDVPHGTPPFC; DLPAVPGPPRPGPAGGALLPRSLTAFVPHSGHGLPV- seq idno 226 -SSGEPAYTPIPHDVPHGTPPFC; QWGLSWMS; seq id no 227 NGDCHGCPEGRQSL;seq id no 228 FTMDRVLTPQWGLSWMS; seq id no 229 FTMDRVLTPPQWGLSWMS; seqid no 230 FTMDRVLTPPNGDCHGCPEGRQSL; seq id no 231FTMDRVLTPNGDCHGCPEGRQSL; seq id no 232HHPARQCPHCIMHLQTQLIHRNLTGPSQLTSLHRS- seq id no 233-PYQIAATPWTTDFAASFFLNPVTPFLLCRRCQGKD- -VLCTNARCLSQTSPSHHKALSRTTTQCMNT--TPWLAVRPAKAFPLL; PHHPARQCPHCIMHLQTQLIHRNLTGPSQLTSLHRS- seq id no 234-PYQIAATPWTTDFAASFFLNPVTPFLLCRRCQGK- -DVLCTNARCLSQTSPSHHKALSRTTTQCMNTTP--WLAVRPAKAFPLL; HTIQHASVPTASCISKLNSYTEN; seq id no 235PQVGMRPSNPPHHPARQCPHCIMHLQTQLIHRNLT- seq id no 236-GPSQLTSLHRSPYQIAATPWTTDFAASFFLNPVTP--FLLCRRCQGKDVLCTNARCLSQTSPSHHKALSRTT- -TQCMNTTPWLAVRPAKAFPLL; PQVGMRPSNPPPHHPARQCPHCIMHLQTQLIHRNL- seq id no 237-TGPSQLTSLHRSPYQIAATPWTTDFAASFFLNPVT--PFLLCRRCQGKDVLCTNARCLSQTSPSHHKALSRT- -TTQCMNTTPWLAVRPAKAFPLL;PQVGMRPSNPPHTIQHASVPTASCISKLNSYTEN; seq id no 238PQVGMRPSNPHTIQHASVPTASCISKLNSYTEN; seq id no 239WAARSWCERRAAAVAPLAPWAWGCPAGCTPPVAARA- seq id no 240 -CAATRPEGWRSPCTH;PWAARSWCERRAAAVAPLAPWAWGCPAGCTPPVAA- seq id no 241 -RACAATRPEGWRSPCTH;RGLRGAGARGGLRLLRHLRPGLGDALRGVHPPLR- seq id no 242-LGPALLPAPRGGEAPAHTDARARRVHGAGGDRGHP- -GPAAL;EEKLARCRPPWAARSWCERRAAAVAPLAPWAWGCPA- seq id no 243-GCTPPVAARACAATRPEGWRSPCTH; EEKLARCRPPPWAARSWCERRAAAVAPLAPWAWGCP- seq idno 244 -AGCTPPVAARACAATRPEGWRSPCTH; EEKLARCRPPRGLRGAGARGGLRLLRHLRPGLGDA-seq id no 245 -LRGVHPPLRLGPALLPAPRGGEAPAHTDARARRVH- -GAGGDRGHPGPAAL;EEKLARCRPRGLRGAGARGGLRLLRHLRPGLGDALR- seq id no 246-GVHPPLRLGPALLPAPRGGEAPAHTDARARRVHGA- -GGDRGHPGPAAL;QPPVSPRPRRPGRPAPPPPQPMVSPRRRTTGPPW- seq id no 247-RPPPLQSTMSPPPQALHQAQLLLWCTTAPLPGLPQ--PQPARALHSQFPATTLILLPPLPAIAPRLMPVALT--IARYLLSPPPITALLPSCLLGSLSFSCLFTFQTSS- -LIPLWKIPAPTTTKSCRETFLKW;SPGCHLGPGDQAAPGLHRPPSPWCHLGAGQQARLGV- seq id no 248-HRPSSPQCHLGLRLCIRLSFYSGAQRHLCQGYHNP- -SQQEHSILNSQPPL;KPAPGSTAPQPPVSPRPRRPGRPRAPPPPQPMVSPR- seq id no 249-RRTTGPPWRPPPLQSTMSPPPQALHQAQLLLWCTT--APLPGLPQPQPARALHSQFPATTLILLPPLPAIAP--RLMPVALTIARYLLSPPPITALLPSCLLGSLSFSC- -LFTFQTSSLIPLWKIPAPTTTKSCRETFLKW;KPAPGSTAPPQPPVSPRPRRPGRPRAPPPPQPMVSP- seq id no 250-RRRTTGPPWRPPPLQSTMSPPPQALHQAQLLLWCT--TAPLPGLPQPQPARALHSQFPATTLILLPPLPAIA--PRLMPVALTIARYLLSPPPITALLPSCLLGSLSFS- -CLFTFQTSSLIPLWKIPAPTTTKSCRETFLKW;KPAPGSTAPPSPGCHLGPGDQAAPGLHRPPSPWCHL- seq id no 251-GAGQQARLGVHRPSSPQCHLGLRLCIRLSFYSGA- -QRHLCQGYHNPSQQEHSILNSQPPL;KPAPGSTAPSPGCHLGPGDQAAPGLHRPPSPWCHL- seq id no 252-GAGQQARLGVHRPSSPQCHLGLRLCIRLSFYSGAQ- -RHLCQGYHNPSQQEHSILNSQPPL;QPMVSPRRRTTGPPWRPPPLQSTMSPPPQALHQAQL- seq id no 253-LLWCTTAPLPGLPQPQPARALHSQFPATTLILLPP--LPAIAPRLMPVALTIARYLLSPPPITALLPSCLLG--SLSFSCLFTFQTSSLIPLWKIPAPTTTKSCRETFL- KW;SPWCHLGAGQQARLGVHRPSSPQCHLGLRLCIRLSF- seq id no 254-YSGAQRHLCQGYHNPSQQEHSILNSQPPL; RPPPGSTAPQPMVSPRRR; seq id no 255RPPPGSTAPPQPMVSPRRR; seq id no 256 RPPPGSTAPPSPWCHLGA; seq id no 257RPPPGSTAPSPWCHLGA; seq id no 258 RPRAPPPPSPWCHL; seq id no 259RPRAPPPPPSPWC; seq id no 260 RPRAPPPPAHGVTSAP; seq id no 261RPRAPPPPPAHGV; seq id no 262 APGLHRPPQPMVSP; seq id no 263AAPGLHRPQPMVSPR; seq id no 264 PGLHRPPPAHGVT; seq id no 265APGLHRPPAHGVTS; seq id no 266 VDRPQHTEWLSWSNLYRIRHQ; seq id no 267HYLCTDVAPR; seq id no 268 HYLCTDVAPPR; seq id no 269HYLCTDVAPPVDRPQHTEWLSWSNLYRIRHQ; seq id no 270HYLCTDVAPVDRPQHTEWLSWSNLYRIRHQ; seq id no 271SAYLSPLGTTWLRTCACRLPRPAASCLCTTPSLLW- seq id no 272-PRRTCPAGSPRATSSPWRMPAPKSCCTTGLAFTS--PIGLGWRSATASGYARIWPVLSLTCQSWSTSLPST- -AVTW;PSAYLSPLGTTWLRTCACRLPRPAASCLCTTPSLLW- seq id no 273-PRRTCPAGSPRATSSPWRMPAPKSCCTTGLAFTSP--IGLGWRSATASGYARIWPVLSLTCQSWSTSLPSTA- -VTW; PAPIFLLWGPLG; seq id no 274APIFLLWGPLG; seq id no 275 LPARAPGPPSAYLSPLGTTWLRTCACRLPRPAASCL- seq idno 276 -CTTPSLLWPRRTCPAGSPRATSSPWRMPAPKSCC--TTGLAFTSPIGLGWRSATASGYARIWPVLSLT- -CQSWSTSLPSTAVTW;LPARAPGPPPSAYLSPLGTTWLRTCACRLPRPAAS- seq id no 277-CLCTTPSLLWPRRTCPAGSPRATSSPWRMPAPKSC--CTTGLAFTSPIGLGWRSATASGYARIWPVLSLTC- -QSWSTSLPSTAVTW;LPARAPGPPPAPIFLLWGPLG; seq id no 278 LPARAPGPPAPIFLLWGPLG; seq id no 279DLEHHGGVTRHRHR; seq id no 280 LVSDYSMTPRP; seq id no 281 LVSDYSMTPPRP;seq id no 282 LVSDYSMTPPDLEHHGGVTRHRHR; seq id no 283LVSDYSMTPDLEHHGGVTRHRHR; seq id no 284FHHIATDVGPFVRIGFLKIKGKIKGKSLRKPNW- seq id no 285 -KTQHKLKRALMFLIVKKL;PFHHIATDVGPFVRIGFLKIKGKIKGKSLRKPNWK- seq id no 286 -TQHKLKRALMFLIVKKL;PSITLQQMLAPS; seq id no 287 SITLQQMLAPS; seq id no 298TSCNEMNPPFHHIATDVGPFVRIGFLKIKGKIKGKS- seq id no 289-LRKPNWKTQHKLKRALMFLIVKKL; TSCNEMNPPPFHHIATDVGPFVRIGFLKIKGKIKG- seq idno 290 -KSLRKPNWKTQHKLKRALMFLIVKKL; TSCNEMNPPSITLQQMLAPS; seq id no 291TSCNEMNPPPSITLQQMLAPS; seq id no 292 LEMILFLMTF seq id no 293HPCITKTFLEMILFLMTF; seq id no 294 HPCITKTFFLEMILFLMTF; seq id no 295HPCITKTFFWR; seq id no 296 HPCITKTFWR; seq id no 297LMFEHSQMRLNSKNAHLPIISF; seq id no 298 EYGSIIAFLMFEHSQMRLNSKNAHLPIISF;seq id no 299 EYGSIIAFFLMFEHSQMRLNSKNAHLPIISF; seq id no 300HLNKGRRLGDKIRAT; seq id no 301 FHLNKGRRLGDKIRAT; seq id no 302VTSGTPFFHLNKGRRLGDKIRAT; seq id no 303 VTSGTPFFFHLNKGRRLGDKIRAT; seq idno 304 VTSGTPFFFI; seq id no 305 VTSGTPFFI; seq id no 306 CEIERIHFFF;seq id no 307 CEIERIHFFSK; seq id no 308 CEIERIHFSK; seq id no 309FRYISKSI; seq id no 310 RYISKSI; seq id no 311 FKKYEPIFFRYISKSI; seq idno 312 FKKYEPIFRYISKSI; seq id no 313FPDSDQPGPLYPLDPSCLISSASNPQELSDCHYIH- seq id no 314-LAFGFSNWRSCPVLPGHCGVQ; PDSDQPGPLYPLDPSCLISSASNPQELSDCHYIHL- seq id no315 -AFGFSNWRSCPVLPGHCGVQ; LNMFASVFS; seq id no 316 LNMFASVFFS; seq idno 317 LNMFASVFFPDSDQPGPLYPLDPSCLISSASNPQE- seq id no 318-LSDCHYIHLAFGFSNWRSCPVLPGHCGVQ; LNMFASVFPDSDQPGPLYPLDPSCLISSASNPQELS-seq id no 319 -DCHYIHLAFGFSNWRSCPVLPGHCGVQ;AMEETVVVAVATVETEVEAMEETGVVAAMEETEVGA- seq id no 320-TEETEVAMEAKWEEETTTEMISATDHT; LWVRPWLWEWLRWRPKWRLWRRQEWWRLWRRPRWGL- seqid no 321 RRRPRWLWRENGRKKRLQK; YGGDRSRGAMEETVVVAVATVETEVEAMEETGVVAA- seqid no 322 -MEETEVGATEETEVAMEAKWEEETTTEMISATDHT;YGGDRSRGGAMEETVVVAVATVETEVEAMEETGVVA- seq id no 323-AMEETEVGATEETEVAMEAKWEEETTTEMISATDH T;YGGDRSRGGLWVRPWLWEWLRWEPKWRLWRRQEWW- seq id no 324-RLWRRPRWGLRRRPRWLWRENGRKKRLQK; YGGDRSRGLWVRPWLWEWLRWEPKWRLWRRQEWWR- seqid no 325 -LWRRPRWGLRRRPRWLWRENGRKKRLQK; EFGGGRRQK; seq id no 326EFGGRRQK; seq id no 327 RRAKGGGAGASNPRQ; seq id no 328 GRRAKGGGAGASNPRQ;seq id no 329 DVGLREGALELPTRGNKRNVA; seq id no 330MRGGGGVGGRRAKGGGAGASNPRQ; seq id no 331 MRGGGGVGGGRRAKGGGAGASNPRQ; seqid no 332 MRGGGGVGGDVGLREGALELPTRGNKRNVA; seq id no 333MRGGGGVGDVGLREGALELPTRGNKRNVA; seq id no 334 VWQLAGPMLAGWRSLGSWFCRMYGI;seq id no 335 CGSWPALCWRAGGVWAVGSAGCMEYDPEALPAAWGP- seq id no 336-AAAATVHPRR; RRYPCEWGVWQLAGPMLAGWRSLGSWFCRMYGI; seq id no 337RRYPCEWGGVWQLAGPMLAGWRSLGSWFCRMYGI; seq id no 338RRYPCEWGGCGSWPAlCWRAGGVWAVGSAGCMEYD- seq id no 339 -EALPAAWGPAAAATVHPRR;RRYPCEWGCGSWPALCWRAGGVWAVGSAGCMEYDPE- seq id no 340 -ALPAAWGPAAAATVHPRR;LWLWAGWTVWWSCGPGEKGHGWPSLPTMALLLLRFS- seq id no 341 -CMRVASY;GLWLWAGWTVWWSCGPGEKGHGWPSLPTMALLLL- seq id no 342 -RFSCMRVASY;GCGCGPAGQYGGAVGLARRGTAGCLPCPPWLCCCCA- seq id no 343-FPACGLPGTDGWRGWQGSGCVRVSGSAPWAPGFPF- -SPPCPLCGTQPRW;CGCGPAGQYGGAVGLARRGTAGCLPCPPWLCCCCAF- seq id no 344-PACGLPGTDGWRGWQGSGCVRVSGSAPWAPGFPFSP -PCPLCGTQPRW;LAFNVPGGLWLWAGWTVWWSCGPGEKGHGWPSLPTM- seq id no 345 -ALLLLRFSCMRVASY;LAFNVPGGGLWLWAGWTVWWSCGPGEKGHGWPSLPT- seq id no 346 -MALLLLRFSCMRVASY;LAFNVPGGGCGCGPAGQYGGAVGLARRGTAGCLPCP- seq id no 347-PWLCCCCAFPACGLPGTDGWRGWQGSGCVRVSGSA- -PWAPGFPFSPPCPLCGTQPRW;LAFNVPGGCGCGPAGQYGGAVGLARRGTAGCLPCPP- seq id no 348-WLCCCCAFPACGLPGTDGWRGWQGSGCVRVSGSAP- -WAPGFPFSPPCPLCGTQPRW;PPMPMPGQREAPGRQEA; seq id no 349 GPPMPMPGQREAPGRQEA; seq id no 350GHQCQCQGKGRHRADRRPDTAQEE; seq id no 351 HQCQCQGKGRHRADRRPDTAQEE; seq idno 352 GGHSYGGGPPMPMPGQREAPGRQEA; seq id no 353GGHSYGGGGPPMPMPGQREAPGRQEA; seq id no 354GGHSYGGGGHQCQCQGKGRHRADRRPDTAQEE; seq id no 355GGHSYGGGHQCQCQGKGRHRADRRPDTAQEE; seq id no 356 APCPQSSGGG; seq id no 357LPAPSQAAADELDRRPG; seq id no 358 TKVRLIRGAPCPQSSGGG; seq id no 359TKVRLIRGGAPCPQSSGGG; seq id no 360 TKVRLIRGGLPAPSQAAADELDRRPG; seq id no361; TKVRLIRGLPAPSQAAADELDRRPG; seq id no 362CSLAKDGSTEDTVSSLCGEEDTEDEELEAAASHLNK- seq id no 363 -DLYRELLGG;GCSLAKDGSTEDTVSSLCGEEDTEDEELEAAASHLN- seq id no 364 -KDLYRELLGG;AAAWQKMAPPRTPRPACVARR; seq id no 365ENSRPKRGGCSLAKDGSTEDTVSSLCGEEDTEDEEL- seq id no 366 -EAAASHLNKDLYRELLGG;ENSRPKRGGGCSLAKDGSTEDTVSSLCGEEDTEDE- seq id no 367-ELEAAASHLNKDLYRELLGG; ENSRPKRGGAAAWQKMAPPRTPRPACVARR; seq id no 368ENSRPKRGAAAWQKMAPPRTPRPACVARR; seq id no 369 HCVLAASGAS; seq iD no 370GHCVLAASGAS; seq id no 371 GTASSRPLGLPKPHLHRPVPIRHPSCPK; seq id no 372TASSRPLGLPKPHLHRPVPIRHPSCPK; seq id no 373 AGTLQLGGHCVLAASGAS; seq id no374 AGTLQLGGGHCVLAASGAS; seq id no 375AGTLQLGGGTASSRPLGLPKPHLHRPVPIRHPSCPK; seq id no 376AGTLQLGGTASSRPLGLPKPHLHRPVPIRHPSCPK; seq id no 377 RRTPSTEKR; seq id no378 RRTPSTEKKR; seq id no 379 RRTPSTEKKGRSEC; seq id no 380RRTPSTEKGRSEC; seq id no 381 STTKCQSGTAETYNSWKVKNLQLEPRRVTSQMNRQV- seqid no 382 -KDMTAILSQS; SSEEIKKKSTTKCQSGTAETYNSWKVKNLQLEPRRV- seq id no384 -TSQMNRQVKDMTAILSQS SSEEIKKKKSTTKCQSGTAETYNSWKVKNLQLEPRR- seq id no385 -VTSQMNRQVKDMTAILSQS; SSEEIKKKKVQPNASQAQQKPTTHGR; seq id no 386SSEEIKKKVQPNASQAQQKPTTHGR; seq id no 387 NRGWVGAGE; seq id no 388; IEAG;seq id no 389 VHNYCNMKNRGWVGAGE; seq id no 390 VHNYCNMKKNRGWVGAGE; seqid no 391 VHNYCNMKKIEAG; seq id no 392 VHNYCNMKIEAG; seq id no 393QLRCWNTWAKMFFMVFLIIWQNTMF; seq id no 394VKKDNHKKQLRCWNTWAKMFFMVFLIIWQNTMF; seq id no 395VKKDNHKKKQLRCWNTWAKMFFMVFLIIWQNTMF; seq id no 396 VKKDNHKKKNS; seq id no397 VKKDNHKKNS; seq id no 398 GAEESGPFNRQVQLKVHASGMGRHLWNCPAFWSEV; seqid no 399 HPSPPPEKRS; seq id no 400 HPSPPPEKKRS; seq id no 401HPSPPPEKKGAEESGPFNRQVQLKVHASGMGRHLW- seq id no 402 -NCPAFWSEV;HPSPPPEKGAEESGPFNRQVQLKVHASGMGRHLWN- seq id no 403 -CPAFWSEV;MQVLSKTHMNLFPQVLLQMFLRGLKRLLQDLEKSKK- seq id no 404 RKL; RCKSARLI; seqid no 405 VQTQPAIKKMQVLSKTHMNLFPQVLLQMFLRGLKRL- seq id no 406-LQDLEKSKKRKL; VQTQPAIKKKMQVLSKTHMNLFPQVLLQMFLRGLKR- seq id no 407-LLQDLEKSKKRKL VQTQPAIKKRCKSARLI; seq id no 408 VQTQPAIKRCKSARLI; seq idno 409 ARSGKKQKRKL; seq id no 410 ARSGKKQKKRKL; seq id no 411ARSGKKQKKENFS; seq id no 412 ARSGKKQKENFS; seq id no 413 KASARSGKSKKRKL;seq id no 414 KASARSGKKSKKRKL; seq id no 415 KASARSGKKAKKENSF; seq id no416 KASARSGKAKKENSF; seq id no 417 HLNKGRRLGDKIRAT; seq id no 418VTSGTPFFHLNKGRRLGDKIRAT; seq id no 419 VTSGTPFFFHLNKGRRLGDKIRAT; seq idno 420 VTSGTPFFFI; seq id no 421 VTSGTPFFI; seq id no 422VTLLYVNTVTLAPNVNMESSRNAHSPATPSAKRK- seq id no 423 -DPDLTWGGFVFFFCQFH;KCRCKPNFFVTLLYVNTVTLAPNVNMESSRNAHSP- seq id no 424-ATPSAKRKDPDLTWGGFVFFFCQFH; KCRCKPNFFFVTLLYVNTVTLAPNVNMESSRNAH- seq idno 425 -SPATPSAKRKDPDLTWGGFVFFFCQFH KCRCKPNFFL; seq id no 426 KCRCKPNFL;seq id no 427 LVKKLKEKKMNWIL; seq id no 429 LVKKLKEKKKMNWIL; seq id no430 LVKKLKEKKR; seq id no 431 LVKKLKEKR; seq id no 432 AAIVKDCCR; seq idno 433 SQPASILGRKL; seq id no 434 SQPASILGKRKL; seq id no 435SQPASILGKAAIVKDCCR; seq id no 436 SQPASILGAAIVKDCCR; seq id no 437NTWAKMFFMVFLIIWQNTMF; seq id no 459

Examples of cancers particularly suitable for treatment with one or acombination of several of this compounds are: colorectal cancer, breastcancer, small-cell lung cancer, non small-cell lung cancer, liver cancer(primary and secondary), renal cancer, melanoma, ovarian cancer, cancerof the brain, head and neck cancer, pancreatic cancer, gastric cancer,eosophageal cancer, prostate cancer and leukemias and lymphomas.

Below are listed some examples of where these mutations may result ingene products that result in development of tumours:

Development of colorectal cancers are believed to result from a seriesof genetic alterations. Deleted in colorectal cancer (DCC) gene (seq idnos 30–34), Human cysteine protease (ICErel-III) gene (seq id nos394–398 and 459), Human putative mismatch repair/binding protein (hMSH3)gene (Seq id nos 134–147), Human hMSH6 gene (seq id nos 200–203 and293–297, Human n-myc gene (seq id nos 189–194), Human TGFβ2 (hTGFβ2)gene (seq id nos 22–29), Human p53 associated gene (seq id nos 285–292may be involved in colorectal cancer.

Human breast cancer susceptibility (BRCA2) (seq id nos 35–94) and HumanBRCA1-associated RING domain protein (BARD1) gene (seq id nos 404–417)are involved in breast cancer and ovarian cancer Human hMSH6 gene (seqid nos 200–203 and 293–297) may be involved in brain tumours.

Gene alteration are frequent in many types of adenocarcinomas, below arelisted some genes that are mutated in many cancers:

Human breast cancer susceptibility (BRCA2) gene (seq id nos 35–94),Deleted in colorectal cancer (DCC) gene (seq id nos 30–34), Humanputatative mismatch repair/binding protein (hMSH3) gene (seq id nos134–147), Human hMSH6 gene (seq id nos 200–203 and 293–297), human N-MYCgene (seq id no 189–194), Human TGFb2 (hTGFb2) gene (seq id nos 22–29),Human p53 associated gene (seq id nos 285–292), Human MUC1 gene (seq idnos 247–266), Human germline n-myc gene (seq id nos 182–188), HumanWilm's tumour (WIT-1) associated protein (seq id nos 388–393), Humannasopharynx carcinoma EBV BNLF-1 gene (seq id nos 204–210), Humantransforming growth factor-beta induced gene product (BIGH3) (seq id nos227–232).

Many of the mutated genes may result in development of leukemias andlymphomas: Human neurofibromin (NF1) gene (seq id nos 176–181), b-rafoncogene (seq id nos 170–175), Human protein-tyrosine kinase (JAK1) gene(seq id nos 267–271), Human protein-tyrosine kinase (JAK3) gene (seq idnos 272–279) are examples.

Genes involved in malignant melanoma: Human malignant melanomametastasis-supressor (hKiSS-1) gene (seq id nos 328–334), Genes involvedin metastasis: Human metastasis-associated mtal (hMTA1) gene (seq id nos357–362).

Cell cycle control and signal transduction is strictly regulated.Frameshift mutations in these genes may result in uncontrolled cellgrowth. Examples of genes which may be susceptible are: Human proteintyrosine phosphatase (hPTP) gene (seq id nos 95–102), Human kinase (TTK)gene (seq id nos 109–120), Human transcriptional repressor (CTCF) gene(seq id nos 121–127), Human cell cycle regulatory protein (E1A-bindingprotein) p300 gene (seq id nos 211–218), Human transforming growthfactor-beta induced gene product (BIGH3) (seq id nos 227–232), HumanFLt4 gene (for transmembrane tyrosinase kinase (seq id nos 280–284),Human G protein-coupled receptor (hGPR1) gene (seq id nos 314–319),Human transcription factor (hITF-2) gene (seq id nos 326–327), Humantelomerase-associated protein TP-1 (hTP-1) gene (seq id nos 335–348),Human transcription factor TFIIB 90 kDa subunit (hTFBIIB90) gene (seq idnos 363–369), Human FADD-homologous ICE/CED-3like protease gene (seq idnos 128–133)

Mutations in DNA synthesis or repair enzymes may also lead touncontrolled cell growth. Human DNA topoisomerase II (top2) gene (seq idnos 103–108) and Human putative mismatch repair/binding protein (hMSH3)gene (seq id nos 134–147) and (hMSH6) gene (seq id nos 200–203 and293–297).

The following are tumour suppressor genes, Human retinoblastoma bindingprotein 1 isoform I (hRBP1) gene (seq id nos 148–156), Humanneurofibromin (NF1) gene (seq id nos 176–181), Human p53 associated gene(seq id nos 285–292, Human retinoblastoma related protein (p107) gene(seq id nos 310–313), Human tumour suppressor (hLUCA-1) gene (seq id nos370–377), Mutations in these genes may result in development of cancer.

The following are oncogenes, proto-oncogenes or putative oncogenes;

Human germline n-myc gene (seq id nos 182–188), Human n-myc gene (seq idnos 189–194), Human can (hCAN) gene (seq id nos 298–300), Human dek(hDEK) gene (seq id nos 307–309), b-raf oncogene (seq id nos 170–175),Human DBL (hDBL) proto-oncogene/Human MCF2PO (hMCF2PO) gene (seq id nos301–306). Frameshift mutations in these genes may lead to development ofcancer.

Biological Experiments

DESCRIPTION OF THE FIGURES

FIG. 1:

It has been demonstrated that T cells from normal donors can bestimulated with a mixture of peptides containing both mutant BAX andmutant TGFβRII peptides. Peptide mixture dependent T cell proliferationin blood samples from six different donors are shown in FIG. 1. Theresults were obtained by stimulating peripheral blood mononuclear cells(PBMCs) from each donor with a mixture of mutant BAX peptides (seq idnos 1,9–12) and mutant TGFβRII peptides (seq id nos 15–21). Theconcentration of each cells were isolated from the cell product beforeDCs were derived using standard methods.

FIG. 4:

FIG. 4 shows the capability of T cells obtained from ascites fluid of apancreatic cancer patient to recognise and proliferate to differentsynthetic peptides derived from mutant BAX (seq id nos 1,9–12) andmutant TGFβRII (seq id nos 15,17–21). The T cell line was obtained afterexpansion of T cells present in the ascites fluid of a patient withpancreatic adenocarcinoma. The T cell line was expanded in vitro byculturing with 100 U/ml recombinant interleukin-2 (rIL-2) (Amersham,Aylesbury, UK) for one week before beeing tested in a proliferationassay.

Autologous, irradiated (30Gy) PBMCs were seeded 5×104 in u-bottomed96-well plates (Costar, Cambridge, Mass.) and pulsed with singlesynthetic peptides at 20 μM for 2 h. The T cells were added 5×104 perwell and the plates were incubated for four days at 37° C. with additionof 18.5×104 Bq/mL 3H-thymidine for the last 12 hours before harvesting.The plates were counted in a liquid scintillation counter (PackardTopcount). Data represent specific proliferation to the differentsynthetic peptides and values are expressed as the mean of triplicatecultures. These results show that T cells isolated from a pancreaticcancer patient are capable of responding to a panel of peptides carryingamino acid sequences derived from mutant BAX and TGFβRII.

FIG. 5:

FIG. 5 further demonstrates the capability T cells from anotherpancreatic cancer patient to recognise and proliferate to differentsynthetic peptides derived from mutant BAX and mutant TGFβRII. The Tcell line was obtained after expansion of T cells present in the ascitesfluid of individual peptide in the mixture was 20 μM. After two weeks,and weekly thereafter, the bulk cultures were restimulated withautologous PBMCs pulsed with 10–25 μM of the peptide mixture. After 4–5restimulations the bulk cultures were tested in a standard proliferationassay with PBMCs alone or as a control or PBMCs pulsed with 25 μM of thepeptides as antigen presenting cells (APCs).

FIG. 2:

It has further been found that T cell clones can be generated againstseparate peptides of the mixture used in the bulk stimulationexperiments. FIG. 2 shows the proliferation of T cell clone 521–2 whichwas obtained by cloning the bulk culture from donor 1 (FIG. 1) byseeding 5 cells per well in U-bottomed, 96-well microtiter plates andusing autologous PBMCs pulsed with 25 μM of the mutant BAX peptide withseq id no 12 as feeder cells. Autologous B-lymphoblastoid cells wereused as APCs in the proliferation assay.

FIG. 3:

In figure three it is shown that mutant BAX peptides and mutant TGFβRIIpeptides can be used to stimulate T cells (PBMCs) from a patient withbreast cancer. Dendritic cells (DCs) from the same cancer patient wereused as APCs. The T cell stimulation (FIG. 3) was obtained by pulsingDCs separately with a mixture of mutant BAX peptides (seq id nos 1,9–12)and a mixture of mutant TGFβRII peptides (seq id nos 15–21) followed byaddition of autologous PBMCs and 10 ng/ml tumour necrosis factor. Theconcentration of each peptide in the mixtures used for pulsing was 25μM. The PBMCs and the DCs were obtained by leukapheresis from a patientwith breast cancer who had been on a granulocyte colony stimulatingfactor (G-CSF) treatment. The CD34+ a patient with pancreaticadencarcinoma. The experiment was set up in the same way as describedabove. Data represent specific proliferation to the different syntheticpeptides and values are expressed as the mean of triplicate cultures.

In order to investigate the T cell response from the latter pancreaticcancer patient, responding T cells were cloned. Peritoneal macrophageswere irradiated (30 Gy) and plated 1×104 into U-bottomed 96-well plates(Costar) together with 25 μM of each peptide. T cell blasts were countedin a microscope and added 5 blasts per well together with 100 U/ml humanrecombinant interleukin-2 (rIL-2) (Amersham, Aylesbury, UK) in a totalvolume of 200 mL. After 14 days T cell clones were transferred onto24-well plates (Costar) with 1 mg/mL phytohemagglutinin (PHA, Wellcome,Dartford, UK), 100 U/ml rIL-2 and allogeneic, irradiated PBMCs as feedercells and screened for peptide specificity after 7 and 14 days.

FIG. 6:

T cell clone 520.5, 520.7 and 520.8 were selected for furthercharacterisation and express the cell surface phenotype CD3+, CD8+ andTcR+. FIG. 6 shows the recognition and cytotoxicity of T cell clone520.5, 520.7 and 520.8 against peptide-pulsed autologous target cellspulsed with the seq id no 10 peptide. Autologous Epstein-barr virustransformed B-cells (EBV) were labelled with 3H-thymidine (9.25×104Bq/ml) over night, washed once and plated 2500 cells per well in 96-wellplates with or without 25 mM of synthetic peptide (seq id no 10) and 1%DMSO in medium. After 30 minutes incubation at 37° C. the plates werewashed before addition of T cells. The plates were further incubated at37° C. for 4 hours and then harvested before counting in a liquidscintillation counter (Packard Topcount). Data represent percentspecific lysis of 3H-thymidine labelled peptide pulsed target cells atan effector/target ratio of 10/1. Values are expressed as the mean oftriplicate cultures. These results demonstrate that the three differentT cell clones obtained from ascites fluid of a pancreatic carcinomapatient, exhibit specific cytotoxicity of autologous EBV targets pulsedwith the relevant peptide (seq id no 10) derived from mutant BAX.

FIG. 7:

FIG. 7 shows the cytolytic properties of three different T cell clonesobtained from the same patient. These T cell clones were cultured andexpanded as described above, but they were generated against a syntheticpeptide the seq id no 17 peptide carrying amino acid sequences derivedfrom mutant TGFβRII. T cell clone 538.1, 538.3 and 538.4 all show thecell-surface phenotype CD3+, CD8+ and TcR+. The experimental conditionswere as described above (FIG. 6). Data represent percent specific lysisof 3H-thymidine labelled peptide pulsed target cells pulsed with the seqid no 428 peptide at an effector/target ratio of 10/1. Values areexpressed as the mean of triplicate cultures. These results demonstratethat the three different T cell clones obtained from ascites fluid of apancreatic carcinoma patient, exhibit specific cytotoxicity ofautologous EBV targets pulsed with the relevant peptide (seq id no 428)derived from mutant TGFβRII.

FIG. 8:

FIG. 8 shows the specificity of two CD4+ T cell clones, IMT8 and IMT9,obtained from a tumour biopsy taken from a patient with anadenocarcinoma localised to the proximal colon. Immunohistochemistryrevealed that the patient had an abundant infiltrate of predominantlyCD4+ T cells, many of which carried activation markers. In areas of CD4T cell infiltration islands of HLA DR positive tumour cells wereobserved. The T cell clones were obtained from the component of tumourinfiltrating lymphocytes which grew out of the biopsy following culturein medium containing 15 U/ml of recombinant human IL-2 for 16 days. TheT cells from this culture were cloned by limiting dilution (1cells/well) in Terasaki plates with irradiated peptide pulsed APC and100 U/ml of IL-2. Pulsing of autologous APC was performed with a mixtureof the TGFβRII frameshift peptides with sequence identity no. 15, 17 and18 at 1 μg/ml of each peptide in the presence of 3 μg/ml of purifiedhuman β2 microglobulin and 10 ng/ml of recombinant human TNFα for 3 hrsat 37° C. Of the 14 clones that could be expanded preliminary testsshowed that two of the clones were reactive with the peptide mixtureused for cloning. After expansion the clones were screened forreactivity with the single peptides in a standard proliferative assay.The results show that IMT8 and IMT9 both react specifically with theTGFβRII frameshift peptide with seq. id. no. 17, no reactivity wasobserved with the two other frameshift peptides tested.

The figure (FIG. 8) depicts the results of conventional T cellproliferative assays, where cloned T cells (5×10⁴) and irradiated APC(5×10⁴) were cocultured for 3 days in triplicates before harvesting. Tomeasure the proliferative capacity of the cultures, ³H-thymidine(3.7×10⁴ Bq/well) was added to the culture overnight before harvesting)Values are given as mean counts per minute (cpm) of the triplicates.

FIG. 9:

FIG. 9 demonstrates that the specific reactivity of the two T cellclones IMT8 and IMT9 against the peptide with seq. id.no. 17 iscompletely blocked by treatment of the cells with an antibody thatspecifically binds to HLA-DR molecules, since the reactivity afterblocking is the same as the background reactivity of the clones with APCin the absence of the peptide. On the other hand antibodies to the HLAclass II isotypes HLA-DQ and -DP failed to block the reactivity of theclones with peptide pulsed APC. This experiment unequivocally identifiesHLA-DR as the molecule responsible to present the peptide to these two Tcell clones. Antibody blocking experiments were performed using thehomozygous EBV transformed cell line 9061 (IHWS9 nomenclature) as APC.The APC were pulsed with peptide at a concentration of 15 μg/1 ml for 1hr at 37° C. before addition of blocking antibodies L243 (pan-DRantibody), SPVL3 (pan-DQ antibody) and B7.21 (pan-DP antibody) at 10μg/ml. Unpulsed APC and APC pulsed with peptide in the absence ofblocking antibody served as negative and positive controls respectively.Results are expressed as in FIG. 8.

FIG. 10:

The patient IMT was HLA typed and turned out to be HLA: A1,2; B7,8;DR3,14; DQ1,2. To determine which of the HLA-DR molecules that wereresponsible for presentation of the peptide with seq. id. no. 17, apanel of HLA workshop derived homozygous BCL cell lines were obtainedand pulsed with the peptide with seq. id. no. 17. FIG. 10 describes theidentification of HLA-DR14 (DRA*0102, DRB*1401) as the HLA-DR moleculeresponsible for presentation of the peptide with seq. id. no. 17 to theT cell clones IMT8 and IMT9. A specific proliferative response wasobserved when peptide was presented by the autologous EBV transformedcell line (Auto APC) and by cell lines 9054 (EK) and 9061 (31227ABO),both of which expressed DR14 as the only DR molecule on their surface.The homozygous cell line gave higher responses, reflecting a higherlevel of expression of the relevant class II/peptide complexes due tothe effect of a double dose of the genes encoding this DR molecule. Noresponse was obtained when the peptide was presented by cell linesexpressing HLA-DR3 (9018, LOO81785), which represents the other DRmolecule expressed by the patients APC, nor by irrelevant HLA-DRmolecules. The experiment was performed as described in FIG. 9, with theexception that no antibody blocking was performed. Results are expressedas in FIG. 8.

FIG. 11:

FIG. 11 describes the dose response curves obtained by pulsing the cellline 9054 with increasing concentrations of the peptide with seq. id.no. 17. Both IMT 8 and IMT9 demonstrate a dose dependent increase in theproliferative response to the peptide. Results were performed asdescribed in FIGS. 9 and 10 with the peptide concentrations indicated onthe Figure (FIG. 11). Results are expressed as in FIG. 8.

FIG. 12:

FIG. 12 describes the reactivity of a cell line generated by in vitrostimulation of T cells isolated from peripheral blood from a healthyblood donor (Donor 2892) by weekly stimulation with irradiatedautologous dendritic cells pulsed with the peptides with sequenceidentity numbers 16, 17 and 21. A specific response above backgroundvalues was obtained when the T cells were co-incubated with autologousdendritic cells pulsed with the peptide with seq. id. no. 21. Noactivity could be detected in the culture after the first and second invitro stimulation. These data demonstrate that the T cell repertoire ofnormal individuals contain a few precursor cells that have the capacityto recognise this frameshift peptide derived from a mutation in TGFβRIIthat does not occur in normal people. In two other blood donors (#2706and #2896), the level of precursor cells with the relevant specificitywas too low to be detected. The results are expressed as spots per 10⁴ Tcells tested in a conventional IFNg ELISPOT assay. This assay enumeratesthe number of cells present in a mixture of cells that are capable ofspecifically reacting with a defined antigen. Briefly 10⁷ T cells (nonadherent cells) were stimulated weekly with 2–5×10⁶ irradiated peptidepulsed autologous dendritic cells (DC) as APC. The DC were generatedfrom the adherent cell population by culture for one week in recombinanthuman GM-CSF and IL-4 according to standard protocols as described inthe literature. After peptide pulsing overnight at 15 μg/ml of peptide,full maturation of the DC was obtained by culture with recombinant TNFα.ELISPOT was performed according to standard published protocols using10⁴ cultured T cells per well in duplicate and 10⁴ peptide pulsed orunpulsed DC as APC. The results are expressed as mean number of spotsper 10⁴ T cells.

FIG. 13:

FIG. 13 shows the results of in vitro stimulation of T cells from ahealthy blood donor (Donor 322) with peptides with sequence identitynumber 15–21. In vitro culture was performed as described in FIG. 12. Aproliferative response above background values was seen when the T cellculture primed with a mixture of the peptides with seq. id. no. 16 and21 was stimulated with peptide 21 and the culture primed with thepeptide with seq. id. no. 17 was stimulated with the same peptide. Theseresults demonstrate that normal blood donors have small numbers ofcirculating T cells specific for these frameshift peptides, and that itis possible to expand these cells in culture by stimulation withframeshift peptides. These results also confirmed the results shown inFIGS. 8–11, demonstrating that the peptide with seq. id. no. 17 isimmunogenic in humans, and indicate that the peptide with seq. id. no.21 may also be used as a cancer vaccine in humans. The results areexpressed as described in FIG. 8.

FIG. 14:

The results shown in FIG. 14 demonstrate that CD8+ T cells specific forHLA class I epitopes can be generated from T cells present in the T cellrepertoire of a healthy blood donor (donor 905). No reactivity abovebackground was seen with any of the peptides after the second round ofin vitro restimulation. After the fourth restimulation, the frequency ofT cells specific for the peptide with seq. id. no. 428 had increasedfrom undetectable levels to approximately 2.5% of the cells. Theseresults demonstrate that CTL precursors of the CD8+ phenotype arepresent in the unprimed T cell repertoire of healthy blood donors. SuchT cells may be expanded in vitro by specific stimulation with thepeptide with seq. id. no. 428. This forms the basis for using thispeptide as a cancer vaccine to elicit cytotoxic T cells specific forframshift peptides in cancer patient having such mutations. T cells weregenerated by weekly restimulation of T cells isolated from peripheralblood and stimulated with peptide pulsed autologous DC as described inFIG. 12, with the exception that Il-7 and Il-2 was added during cultureaccording to standard procedures for generating cytotoxic T cells (CTL)of the CD8 phenotype. The peptides used were peptides with sequenceidentity number 428, 439, 446 and 451. Cells were tested in ELISPOTassay as described in FIG. 12. The results are expressed as described inFIG. 12.

The peptide with seq. id. no. 17 was selected and designed to containbinding motifs for both several HLA class I and HLA class II molecules.These peptides thus contains epitopes both for CD4+ and CD8+ T cells,and was predicted to elicit both CD4 and CD8 T cell responses in cancerpatient provided processing of the aberrant TGFβRII protein naturallyoccurring in cancer cells would take place and result in an overlappingpeptide. This has now been proven for CD4 T cells by the results inFIGS. 8–11. These results have the following implication:

1) The results in FIG. 8 prove that the mutated form of TGFβRII Receptorwhich occurs in a high proportion of cancer patients with defects intheir mismatch repair machinery is a tumour specific antigen.

2) The antigen specificity of the infiltrating T cells commonly observedin colorectal cancer are generally not known. The results in FIG. 8demonstrate that one component of the T cells constituting thepopulation of tumour infiltrating lymphocytes in this patients tumour isspecific for a frameshift mutation, demonstrating that TGFβRIIframeshift peptides are immunogenic in vivo, occasionally giving rise tospontaneous T cell activation.

3) It follows from this observation that processing of thenon-functional form of the TGFβRII Receptor that is formed by the commonframeshift mutation is processed. This processing may take place eitherin the tumour cell as part of natural breakdown of the aberrant protein,or after the tumour cell itself or a released form of the receptor hasbeen taken up by a professional APC or both.

4) The results in FIG. 8 also indicate that the peptide with seq. id.no. 17 is capable of binding to an HLA class II molecule, since pulsingof APC with this peptide results in a specific proliferative responseagainst the peptide, and since CD4 T cell responses always are class IIrestricted. That this is the case is demonstrated by the results of theexperiment shown in FIG. 9. Here it is shown that the specific responseagainst the peptide with seq. id. no. 17 is completely blocked by anantibody to HLA-DR, but not with antibodies to the two other HLA classII molecules, HLA-DQ and -DP. Furthermore, by using a panel of standardhomozygous Epstein Barr Virus (EBV) transformed B Cell Lines (BCL)covering the relevant HLA class II molecules present on the patients ownAPC, we were able to identify the class II molecule responsible forpresentation of the peptide with seq. id. no. 17 to TLC IMT8 and IMT9 asbeing HLA-DR 14. Together these findings fit extremely well with theimmunohistological observations made in parallel sections taken from thesame tumour biopsy, where we could show that activated CD4+ T cells wereabundant in the proximity of tumour cells that had been induced toexpress HLA-DR. molecules. The results in FIG. 11 demonstrate that theseT cell clones are capable of mounting a proliferative response over arange of peptide doses and that the responses are dose dependent.

5) Since these T cell clones were obtained by cloning T cells isolatedfrom a tumour biopsy, another implication of our finding is thatactivated T cells specific for the peptide with seq. id. no. 17 arecapable of homing to the tumour tissue after activation.

6) Since the peptide with seq. id. no. 17 is a tumour specific antigen,and since frameshift mutations giving rise to this peptide or peptideswith overlapping sequences are commonly found in cancers with defects inenzymes that are part of the mismatch repair machinery, this peptide maybe used as a vaccine to elicit T cell response in cancer patients orpatients at high risk for developing cancer. Such T cell responses maypotentially influence the growth of an existing tumour or prohibitregrowth of tumour after surgery and other forms of treatment or begiven to patients with an inheritable form of cancer where a defectmismatch enzyme is detected or suspected and that have a high chance ofdeveloping a cancer where this precise mismatch repair mutation willoccur.

Synthesis

The peptides were synthesised by using continuous flow solid phasepeptide synthesis. N-a-Fmoc-amino acids with appropriate side chainprotection were used. The Fmoc-amino acids were activated for couplingas pentafluorophenyl esters or by using either TBTU or diisopropylcarbodiimide activation prior to coupling. 20% piperidine in DMF wasused for selective removal of Fmoc after each coupling. Cleavage fromthe resin and final removal of side chain protection was performed by95% TFA containing appropriate scavengers. The peptides were purifiedand analysed by reversed phase (C18) HPLC. The identity of the peptideswas confirmed by using electro-spray mass spectroscopy (Finnigan matSSQ710).

The peptides used for in vitro studies of T cell stimulation weresynthesised by this method.

Several other well known methods can be applied by a person skilled inthe art to synthesise the peptides.

Examples of the Method for Determining New Frameshift Mutation Peptides.

In this Example, the BAX gene is used to illustrate the principle.

In each of the steps listed below, the 1st line is the gene sequence and2nd line is amino acid sequence.

In the steps 2–5, the outlined sequences represent the mutant part ofthe protein.

Step One:

Normal BAX.

AT G GGG GGG G AG GCA CCC GAG CTG GCC CTG GAC CCG    M   G   G   E   A   P   E   L   A   L   D GTG . . . P   V . . .Step Two:

1G deleted from gene sequence.

ATG GGG GGG

 

 

 

 

 

 

 

 

    M   G   G    R   H   P   S   W   P   W   T

 

   

 

 

 

 

 

 

  TGA  R   C  L  R   M   R   P   P   R   S  stopStep Three:

2G deleted from gene sequence.

ATG GGG

 

 

 

 

 

 

 

 

 

 M   G   G   G   T   R   A   G   P   G   P   G

 

 

 

 

 

 

 

 

 

 

 

 A   S   G   C   V   H   Q   E   A   E   R   V

 

 

 

 

 

 

 

 

 

  TAA  S   Q   A   H   R   G   R   T   G   Q  stopStep Four:

1G inserted in gene sequence.

ATG GGG GGG

 

 

 

 

 

 

 

 

 M   G   G   G   G   T   R   A   G   P   G   P

 

 

 

 

 

 

 

 

 

 

 

 G   A   S   G   C   V   H   Q   E   A   E   R

 

 

 

 

 

 

 

 

 

 

  TAA  V   S   Q   A   H   R   G   R   T   G   Q  stopStep Five:

2G inserted in gene sequence.

ATG GGG GGG GGG

 CAC CCG AGC TGG CCC TGG ACC M   G   G   G   R   H   P   S   W   P   W   T CGG TGC CTC AGG ATG CGTCCA CCA AGA AGC TGA  R   C   L   R   M   R   P   P   R   S  stop

In the next Example, the TGFβRII gene is used to illustrate theprinciple.

In each of the steps listed below, the 1st line is the gene sequence and2nd line is amino acid sequence.

In the steps 2–5, the outlined sequences represent the mutant part ofthe protein.

Step One:

Normal TGFβRII.

G AA AAA AAA AA G CCT GGT GAG ACT TTC TTC ATG TGT    E   K   K   K   P   G   E   T   F   F   M TCC . . . C   S . . .Step Two:

1A deleted from gene sequence.

G

 

 

 

 

 

 

 

 

 E   K   K   S   L   V   R   L   S   S   C   V

 

 

 

 

 

 

 

 

 

 

 

 P   V   A   L   M   S   A   M   T   T   S   S

 

 

 

 

 

 

 

 

 

 

 

 S   Q   K   N   I   T   P   A   I   L   T   C

  TAG  C  stopStep Three:

2A deleted from gene sequence.

G AA AAA AAA  

 

  TGA  E   K   K   A   W  stopStep Four:

1A inserted in gene sequence.

G

 

 

  TGA  E   K   K   K   A   W  stopStep Five:

2A inserted in gene sequence.

G

 

 

 

 

 

 

 

 E   K   K   K   S   L   V   R   L   S   S   C

 

 

 

 

 

 

 

 

 

 

 

 V   P   V   A   L   M   S   A   M   T   T   S

 

 

 

 

 

 

 

 

 

 

 

 S   S   Q   K   N   I   T   P   A   I   L   T

 

  TAG  C   C  stop

Thus the peptides of the invention may be used in a method for thetreatment of cancers with cancer cells harbouring genes with frameshiftmutations, which treatment comprises administering at least one peptideof the present invention in vivo or ex vivo to a human patient in needof such treatment.

In another embodiment the peptides of the invention may be used tovaccinate a human being disposed for cancers with cancer cellsharbouring genes with frameshift mutations, by administering at leastone peptide of the present invention to said human being.

It is further considered to be an advantage to administer to a humanindividual a mixture of the peptides of this invention, whereby each ofthe peptides of the invention can bind to different types of HLA class Iand/or class II molecules of the individual.

It is further anticipated that the power of an anticancer vaccine orpeptide drug as disclosed in the above mentioned PCT/NO92/00032application, can be greatly enhanced if the peptides of the presentinvention were included. Thus in another embodiment of the presentinvention peptides of the present invention are administered togetherwith, either simultaneously or in optional sequence, with the peptidesdisclosed in PCT/NO92/00032.

It is considered that the peptides may be administered together, eithersimultaneously or separately, with compounds such as cytokines and/orgrowth factors, i.e. interleukin-2 (IL-2), interleukin-12 (IL-12),granulocyte macrophage colony stimulating factor (GM-CSF), Flt-3 ligandor the like in order to strengthen the immune response as known in theart.

The peptides according to the present invention can be used in a vaccineor a therapeutical composition either alone or in combination with othermaterials, such as for instance standard adjuvants or in the form of alipopeptide conjugate which as known in the art can induce high-affinitycytotoxic T lymphocytes, (K. Deres, Nature, Vol. 342, (November 1989)).

The peptides according to the present invention may be useful to includein either a peptide or recombinant fragment based vaccine.

The peptides according to the present invention can be included inpharmaceutical compositions or in vaccines together with usualadditives, diluents, stabilisers or the like as known in the art.

According to this invention, a pharmaceutical composition or vaccine mayinclude the peptides alone or in combination with at least onepharmaceutically acceptable carrier or diluent.

Further a vaccine or therapeutical composition can comprise a selectionof peptides which are fragments of the mutant proteins arising frominsertion or deletion of bases in a repeat sequence of the gene.

Further a vaccine composition can comprise at least one peptide selectedfor one cancer, which vaccine would be administered to a person carryinga genetic disposition for this particular cancer.

Further a vaccine composition can comprise at least one peptide selectedfor one cancer, which vaccine would be administered to a personbelonging to a high risk group for this particular cancer.

The cancer vaccine according to this invention may further beadministered to the population in general for example as a mixture ofpeptides giving rise to T cell immunity against various common cancersconnected with frameshift mutation genes.

The peptides according to this invention may be administered as singlepeptides or as a mixture of peptides. Alternatively the peptides may becovalently linked with each other to form larger polypeptides or evencyclic polypeptides.

A cancer therapy according to the present invention may be administeredboth in vivo or ex vivo having as the main goal the raising of specificT cell lines or clones against the mutant gene product associated withthe cancer type with which the patient is afflicted.

Further, the frameshift mutant peptides of this invention may beadministered to a patient by various routes including but not limited tosubcutaneous, intramuscular, intradermal, intraperitoneal, intravenousor the like. In one embodiment the peptides of this invention areadministered intradermally. The peptides may be administered at singleor multiple injection sites to a patient in a therapeutically orprophylactically effective amount.

The peptides of this invention may be administered only once oralternatively several times, for instance once a week over a period of1–2 months with a repeated sequence later all according to the need ofthe patient being treated.

The peptides of this invention can be administered in an amount in therange of 1 microgram (1 μg) to 1 gram (1 g) to an average human patientor individual to be vaccinated. It is preferred to use a smaller dose inthe rage of 1 microgram (1 μg) to 1 milligram (1 mg) for eachadministration.

The invention further encompasses DNA sequences which encodes aframeshift mutation peptide.

The invention additionally encompasses isolated DNA sequences comprisinga DNA sequence encoding at least one frameshift mutant peptide, andadministration of such isolated DNA sequences as a vaccine for treatmentor prophylaxis of cancers associated with frameshift mutations in thegenes.

The peptides according to this invention may be administered to anindividual in the form of DNA vaccines. The DNA encoding these peptidesmay be in the form of cloned plasmid DNA or synthetic oligonucleotide.The DNA may be delivered together with cytokines, such as IL-2, and/orother co-stimulatory molecules. The cytokines and/or co-stimulatorymolecules may themselves be delivered in the form of plasmid oroligonucleotide DNA. The response to a DNA vaccine has been shown to beincreased by the presence of immunostimulatory DNA sequences (ISS).These can take the form of hexameric motifs containing methylated CpG,according to the formula: 5′-purine-purine-CG-pyrimidine-pyrimidine-3′.Our DNA vaccines may therefore incorporate these or other ISS, in theDNA encoding the peptides, in the DNA encoding the cytokine or otherco-stimulatory molecules, or in both. A review of the advantages of DNAvaccination is provided by Tighe et al (1998, Immunology Today, 19 (2),89–97).

In one embodiment, the DNA sequence encoding the mutant BAX peptidescomprises:

Normal BAX. AT G GGG GGG G AG GCA CCC GAG CTG GCC CTG GAC CCG GTG . . .1G deleted from BAX gene sequence. ATG GGG GGG

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  TGAl 2G deleted from BAX gene sequence. ATG GGG

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  TAA 1G inserted in BAX gene sequence. ATG GGG GGG

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  TAA 2G inserted in BAX gene sequence. ATG GGG GGG GGG

 CAC CCG AGC TGG CCC TGG ACC CGG TGC CTC AGG ATG CGT CCA CCA AGA AGC TGA

In a second embodiment, the DNA sequence encoding the mutant TGFβRIIpeptides comprises:

Normal TGFβRII gene. G AA AAA AAA AA G CCT GGT GAG ACT TTC TTC ATG TGTTCC . . . 1A deleted from TGFβRII gene sequence. G

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  TAG 2A deleted from TGFβRII gene sequence. G AA AAA AAA  

 

  TGA 1A inserted in TGFβRII gene sequence. G

 

 

  TGA 2A inserted in TGFβRII gene sequence. G

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  TAG

The invention further encompasses vectors and plasmids comprising a DNAsequence encoding a frameshift mutant peptide. The vectors include, butare not limited to E. Coli plasmid, a Listeria vector and recombinantviral vectors. Recombinant viral vectors include, but are not limited toorthopox virus, canary virus, capripox virus, suipox virus, vaccinia,baculovirus, human adenovirus, SV40, bovine papilloma virus and the likecomprising the DNA sequence encoding a frameshift mutant peptide.

It is considered that an anticancer treatment or prophylaxis may beachieved also through the administration of an effective amount of arecombinant virus vector or plasmid comprising at least one insertionsite containing a DNA sequence encoding a frameshift mutant peptide to apatient, whereby the patient's antigen presenting cells are turned intohost cells for the vector/plasmid and presentation of HLA/frameshiftmutation peptide complex is achieved.

A person skilled in the art will find other possible use combinationswith the peptides of this invention, and these are meant to beencompassed by the present claim.

The peptides according to this invention may be produced by conventionalprocesses as known in the art, such as chemical peptide synthesis,recombinant DNA technology or protease cleavage of a protein or peptideencoded by a frameshift mutated gene. One method for chemical synthesisis elucidated in the description below.

In order for a cancer vaccine and methods for specific cancer therapybased on specific T cell immunity to be effective, three conditions mustbe met:

-   1. The peptides used must correspond, either in their full length or    after processing by antigen presenting cells, to the processed    mutant protein fragment as presented by a HLA Class I and/or class    II molecule on the cancer cell or other antigen presenting cells,-   2. The peptides used must be bound to a HLA Class I and/or Class II    molecule in an immunogenic form, and-   3. T-cells capable of recognising and responding to the HLA/peptide    complex must be present in the circulation of the human being.

It has been established that all these conditions are met for somerepresentative peptides according to the present invention. The peptidesaccording to the present invention give rise to specific T cell immuneresponses in vitro. It has been established that the peptides accordingto this invention correspond to processed mutant protein fragments. Thisis exemplified with peptides corresponding to fragments of transformedmutant BAX and TGFβRII peptides.

Through the present invention the following advantages are achieved:

-   -   It offers a possibility to treat patients suffering from cancers        arising from frame-shift mutations in their genes, most of which        cancers known at present do not have any good treatment        alternatives.    -   It offers a possibility to vaccinate prophylaxtically humans        carrying genetic dispositions or belonging to other high risk        groups.    -   It offers a possibility to prepare a combination treatment for a        specific cancer, such as for instance colorectal or pancreatic        cancers, wherein the cancer commonly is associated with either a        frameshift mutation or a point mutation in the genes.    -   Since described frameshift mutations occurs in a large variety        of cancers it will be possible to use this peptides in        combination with established vaccines and future vaccines to        obtain a multiple targetting treatment.    -   Likewise patients suffering from cancers associated with        multiple frameshift mutations in genes can be treated more        efficiently through a combination treatment.

1. An isolated BAX gene frameshift-mutation peptide selected from thegroup consisting of Seq ID No. 1, Seq ID No. 5, and Seq ID No.
 9. 2. Acomposition comprising a peptide according to claim 1 and a carrier ordiluent therefor.
 3. An isolated peptide consisting of Seq ID No.
 1. 4.An isolated peptide consisting of Seq ID No.
 5. 5. An isolated peptideconsisting of Seq ID No.
 9. 6. A composition comprising a peptideaccording to claim 3 and a carrier or diluent therefor.
 7. A compositioncomprising a peptide according to claim 4 and a carrier or diluenttherefor.
 8. A composition comprising a peptide according to claim 5 anda carrier or diluent therefor.
 9. A method of stimulating theproliferation of human T cells, comprising the steps of: i) obtaining Tcells from a human cancer patient and ii) contacting the T cellsobtained in step i) with a BAX gene frameshift-mutation peptide selectedfrom the group consisting of Seq ID No. 1, Seq ID No. 5, and Seq ID No.9, said peptide being capable of inducing T cell proliferation, eitherin its full length form or after processing by an antigen-presentingcell.
 10. The method according to claim 9, wherein the peptide used instep ii) is Seq ID No.
 1. 11. The method according to claim 9, whereinthe peptide used in step ii) is Seq ID No.
 5. 12. The method accordingto claim 9, wherein the peptide used in step ii) is Seq ID No. 9.